Prediction of muscle performance during dynamic repetitive movement.

BACKGROUND: During long-duration spaceflight, astronauts experience progressive muscle atrophy and often perform strenuous extravehicular activities. Post-flight, there is a lengthy recovery period with an increased risk for injury. Currently, there is a critical need for an enabling tool to optimize muscle performance and to minimize the risk of injury to astronauts while on-orbit and during post-flight recovery. Consequently, these studies were performed to develop a method to address this need. METHODS: Eight test subjects performed a repetitive dynamic exercise to failure at 65% of their upper torso weight using a Lordex spinal machine. Surface electromyography (SEMG) data was collected from the erector spinae back muscle. The SEMG data was evaluated using a 5th order autoregressive (AR) model and linear regression analysis. RESULTS: The best predictor found was an AR parameter, the mean average magnitude of AR poles, with r = 0.75 and p = 0.03. This parameter can predict performance to failure as early as the second repetition of the exercise. CONCLUSION: A method for predicting human muscle performance early during dynamic repetitive exercise was developed. The capability to predict performance to failure has many potential applications to the space program including evaluating countermeasure effectiveness on-orbit, optimizing post-flight recovery, and potential future real-time monitoring capability during extravehicular activity.

Etoposide-resistance in the multidrug-resistant LZ-8 cells.

The multidrug-resistant LZ-8 cells were found to exhibit marked resistance to etoposide compared to wild-type, parental V79 cells. The multidrug resistant phenotype did not significantly contribute to this etoposide-resistance. Following exposure of LZ-8 cells and V79 cells to equivalent concentrations of etoposide, there was a dramatic reduction in the number of etoposide-induced stabilized DNA-topoisomerase II complexes in the LZ-8 cells compared to V79 cells, however, this reduction was not found when nuclei isolated from LZ-8 and V79 cells were exposed to equivalent concentrations of etoposide. These results suggest that cytoplasmic factors are involved in the etoposide-resistance of LZ-8 cells.

Sequestration of doxorubicin in vesicles in a multidrug-resistant cell line (LZ-100).

A multidrug-resistant Chinese hamster cell line, LZ-8, was subcultured in increasing levels of doxorubicin (DOX) until capable of growth in 100 micrograms/mL DOX. This new derivative, designated LZ-100, is the most DOX-resistant line in the LZ series, based on a comparison of Ki-1 values from cell survival studies. This increased level of drug resistance in LZ-100 cells did not result from (i) higher levels of P-glycoprotein (P-gp) in the plasma membrane compared with LZ-8 cells, since this protein constitutes approximately 20% of the total plasma membrane protein in both cell lines, or (ii) more efficient drug pumping by the same amount of P-gp, since efflux of rhodamine 123 and DOX was comparable in the two cell lines. However, an altered drug distribution was observed in LZ-100 cells compared with wild-type V79 cells; in LZ-100 cells DOX was largely excluded from the nucleus and was sequestered in vesicles in the cytoplasm. The number of vesicles per cell seen after DOX exposure corresponded with the level of drug resistance achieved by the LZ cell lines studied. DOX concentration-response experiments revealed that vesicle formation exhibited a biphasic relationship, with an initial rapid increase followed by a plateau where no further increase was observed. Time-course studies in LZ-100 cells revealed that the maximum number of DOX-containing vesicles per cell occurred 3-4 hr following initiation of DOX treatment. Radiation exposure (10 Gy) immediately preceding DOX treatment decreased the number of vesicles formed in LZ-100 cells by more than one-half and altered the subcellular distribution of DOX from an almost exclusively cytoplasmic to a homogeneous nuclear/cytoplasmic distribution. This redistribution was not a result of radiation inhibition of P-gp efflux. The inhibitory effect of radiation on vesicle formation increased with increasing radiation dose up to 10 Gy. Drug-containing vesicles were also observed in LZ-100 cells following exposure to mitoxantrone or daunorubicin (to which LZ-100 cells are also resistant), but fewer vesicles were observed than with DOX. These studies demonstrate that the drug sequestration phenomenon (i) occurs in cells exhibiting widely different levels of drug resistance, (ii) correlates with the level of drug resistance in LZ cell lines, (iii) occurs rapidly following exposure to DOX, mitoxantrone, or daunorubicin, and (iv) can be inhibited by irradiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of chromosome microdissection, the polymerase chain reaction, and dot blot hybridization to analyze double minute chromosomes.

The potential usefulness of chromosome microdissection, the polymerase chain reaction (PCR), and dot blot hybridization as a quick screening method for determining the genetic composition of double minute chromosomes (DMs) was evaluated. DMs or abnormally banding regions (ABRs) were microdissected from multidrug-resistant hamster cell lines and amplified with PCR using primers specific for the hamster multidrug-resistance (MDR) gene, pgp 1. The microdissected-PCR-amplified products were shown to (a) hybridize to a 32P-labeled pCHP1 probe for the hamster MDR gene by using dot blot or Southern blot analysis and also (b) hybridize back to the chromosome region from which they were originally dissected by using fluorescent in situ hybridization. Microdissected/PCR-amplified DMs were also shown to hybridize to ABRs. When microdissected DMs and ABRs were amplified using hamster specific Alu primers, the resulting material was shown to hybridize with probes for hamster MDR and Alu. These results suggest that the DMs contained in these MDR hamster cell lines contain Alu-like sequences and the chromosome microdissection-PCR-hybridization approach might be used as a quick screening method for identifying genes amplified in DMs and ABRs in cell lines and human tumor samples.

Characterization of adriamycin-resistant and radiation-sensitive Chinese hamster cell lines.

A series of cell lines derived from Chinese hamster V79 cells by selection in increasing concentrations of Adriamycin (ADRM) was developed to study the mechanisms of drug resistance and its relationship to radiation response. Survival studies revealed that selection in increasingly higher concentrations of ADRM positively correlated with increased cellular drug resistance. Increased cellular resistance correlated positively with amplification of the hamster multidrug-resistance gene (pgp 1) as detected with dot blot analysis using the pCHP1 probe. Southern blot analysis of restriction endonuclease digested DNA (Eco RI, Hind III, Pst I, or Bam HI) showed that (1) some fragments were preferentially amplified compared to others in the ADRM-resistant lines; and (2) no major gene rearrangement appeared to have occurred during the selection for greater ADRM resistance. Levels of pgp 1 gene expression assayed with dot blot and Northern analysis showed a parallel increase of mRNA with gene amplification and increased ADRM resistance. The amounts of the pgp 1 gene product, P-glycoprotein (P-gp), in the cell membrane of the ADRM-resistant cells correlated with the amount of gene amplification/expression. However, levels of P-gp only correlated with degree of drug resistance as measured by cell survival in earlier selection stages (77A and LZ-3). In later selection stages (LZ-8 and LZ-24), higher levels of ADRM resistance were achieved but levels of P-gp did not increase beyond approximately 20% of plasma membrane proteins. These results suggest that (1) the LZ cell plasma membrane may have a physical limit as to the amount of P-gp it can accommodate and/or there is a cellular mechanism for regulating the amount of P-gp in the plasma membrane, and (2) additional resistance mechanisms are present in LZ-8 and LZ-24 cells. Microscopic observations of intracellular drug distribution in these cell lines revealed that (1) ADRM appeared to be sequestered in cytoplasmic vesicles, and (2) the amount of sequestration (number of vesicles) exhibited correlated with the degree of drug resistance attained by the cell lines. These results suggest that drug sequestration is another mechanism of resistance in LZ cells in addition to P-gp-mediated drug efflux.

An enhanced ability for transforming adriamycin into a noncytotoxic form in a multidrug-resistant cell line (LZ-8).

Multidrug-resistant LZ-8 cells are 9000-fold more resistant to Adriamycin (ADRM) exposure than wild-type V79 cells. To understand more about the mechanisms producing such high level resistance, we tested whether LZ-8 cells inactivate ADRM toxicity to a greater extent than wild-type V79 cells. ADRM was recovered from (1) culture media of wild-type V79 and ADRM-resistant LZ-8 cells; (2) V79 and LZ-8 cells; and (3) LZ-8 cell plasma membrane, and the cytotoxicity was determined by treating V79 cells for 1 hr with a known concentration of the recovered ADRM. ADRM obtained from LZ-8 cells or its culture medium exhibited less cytotoxicity than that recovered from V79 cells or its culture medium. ADRM extracted from LZ-8 cell plasma membrane was noncytotoxic. HPLC analysis revealed that the extracted ADRM was structurally changed compared to stock ADRM. The retention time in the column was 7 min for stock ADRM, and 23 min for the recovered ADRM. Thus, LZ-8 cells have an increased ability to transform ADRM into a noncytotoxic form compared to wild-type V79 cells. This transformation involves structural conversion into a previously unidentified ADRM metabolite. The greatly increased survival of LZ-8 cells compared to V79 cells after ADRM treatment is due to at least two mechanisms: (1) an enhanced ability to inactivate the cytotoxicity of ADRM, and (2) increased drug efflux resulting from the amplification and overexpression of the pgp 1 gene in these cells. Our results suggest the possibility that P-glycoprotein participates in drug binding/inactivation in addition to serving as a drug efflux pump.

Interaction between radiation and drug damage in mammalian cells. VI. Radiation and doxorubicin age-response function of doxorubicin-sensitive and -resistant Chinese hamster cells.

A comparative study of the radiation and/or doxorubicin (DOX) survival response for synchronous populations of Chinese hamster V79 cells and two DOX-resistant variants (77A and LZ-8) was performed. The greatest cellular radiation sensitivity was observed in mitosis, while the greatest resistance was observed during late S phase for the three cell lines. The variation in radiation response throughout the cell cycle was expressed as a change in the width of the shoulder of the survival curves (Dq) with little change in D0. This suggests that each phase of the cell cycle has a different capacity for accumulation of radiation injury. The radiation age-response function for the three cell lines revealed that 77A and LZ-8 cells were more radiosensitive than V79 cells throughout the cell cycle. Exposure of synchronous populations to DOX (1.84 microM for V79, 9.21 microM for 77A, and 921 microM for LZ-8) for 1 h as a function of cell cycle phase revealed that V79, 77A, and LZ-8 cells exhibited the greatest sensitivity to DOX in mitosis and the most resistance to DOX during S phase, as indicated by the differences in the slope of the initial component of the survival curve. Levels of P-glyco-protein (P-gp) are probably not a factor contributing to DOX age-response function since P-gp levels remain constant throughout the cell cycle in all three cell lines. Synchronous populations of V79, 77A, and LZ-8 cells sequentially treated with DOX and radiation at various cell cycle phases were also analyzed. The results showed that the interaction between radiation and DOX damage resulted in a reduced cellular capacity for the accumulation of radiation damage throughout the cell cycle, as indicated by a decrease in the width of the shoulder of the survival curve. Overall, both DOX-sensitive V79 cells and DOX-resistant 77A and LZ-8 cells exhibited (1) a similar age-response function for radiation or DOX, and (2) no differences in the effects of DOX on radiation-induced damage throughout the cell cycle. These results indicate that acquired resistance to DOX associated with increased levels of P-gp in the cell membrane did not appear to affect the age-response function for radiation or DOX, and the nature of the interaction between damage caused by radiation and DOX was also not affected.

Dot-blot hybridization: quantitative analysis with direct beta counting.

The suitability of using direct beta counting (DBC) for quantitating radioactivity of the probe:target complex in dot-blot hybridization was evaluated using a Packard Matrix 96. A comparison of blots analyzed using autoradiography followed by densitometry scanning (film/densitometry) with those analyzed using direct beta counting revealed similar data trends with the two methods. However, direct beta counting quantitated the amount of radioactivity in the dot blots directly (without film exposure or additional sample preparation), which significantly reduced the time required to obtain results. Blots analyzed first with direct beta counting and then liquid scintillation counting exhibited similar data trends with both methods. Despite a decreased counting efficiency, analysis with direct beta counting has the following advantages compared with liquid scintillation counting: 1) no additional sample preparation is required (no vials or cocktail are used), 2) no sample destruction occurs due to analysis and 3) quantitative results are obtained more rapidly (since the radioactivity for all 96 samples in a dot blot is simultaneously determined in real time). Analysis with direct beta counting was also shown not to interfere with the successful reprobing of stripped dot blots with either unique sequence or total genomic probes. Overall, direct beta counting provides quick, quantitative results for dot blots while saving considerable time and effort.

Interaction between radiation and drug damage in mammalian cells. V. DNA damage and repair induced in LZ cells by adriamycin and/or radiation.

A multi-drug-resistant cell line selected in increasing concentrations of Adriamycin and designated LZ (J. A. Belli, Radiat. Res. 119, 88-100, 1989) is shown to exhibit a survival response characterized by radiation sensitivity and Adriamycin resistance. To determine if this response is due to alterations in either the initial levels of damage induced or the repair of DNA damage, LZ cells and the parental V79 cells were exposed to either radiation or Adriamycin and the damage and repair were measured with alkaline or nondenaturing filter elution. After exposure to radiation, induction and repair of both single-strand and double-strand breaks were equivalent. LZ cells exposed to 100 micrograms/ml Adriamycin for 1 h contained no measurable damage while the same treatment induced breaks and crosslinks in V79 cells. Pretreatment of LZ cells for 1 h with Adriamycin before irradiation did not alter either the initial levels of induced damage or the repair of strand breakage. These results suggest that (1) mechanisms other than differential induction and repair of strand breaks are responsible for the increased radiation sensitivity in LZ, and (2) the lack of Adriamycin-induced DNA damage in LZ is at least partially responsible for the increased cell survival after treatment.

Molecular analysis of reactive oxygen-species-induced mammalian gene mutation.

We have studied the mutagenicity and toxicity of physical and chemical agents in the Chinese hamster ovary (CHO) cell line K1-BH4 and its transformant, AS52. The AS52 cells lack the normal X-linked mammalian hypoxanthine-guanine phosphoribosyltransferase (hprt) gene but instead contain a single autosomally integrated copy of the bacterial equivalent, the xanthine-guanine phosphoribosyltransferase (gpt) gene. We found that X-rays and neutrons appear to be equitoxic to both cell types; however, these physical agents are approximately 10 times more mutagenic to the gpt gene of AS52 cells than to the hprt gene of K1-BH4 cells. We reasoned that if reactive oxygens were to mediate the mutagenic effects of both radiomimetic chemicals and radiation, then reactive oxygen-producing chemicals, such as streptonigrin and bleomycin, and oxidizing agents such as potassium superoxide and hydrogen peroxide, would exhibit similar levels of toxicity but different frequencies of mutants when assayed with the two cell lines. Our experiments fulfill such predictions. We postulate that the apparent hypermutability of AS52 cells probably results from a higher recovery of multi-locus deletion mutants in AS52 cells than in K1-BH4 cells, rather than a higher yield of induced mutants. Preliminary studies, using Southern blot and the polymerase chain reaction to analyze the mutational spectrum of the mutants, support our hypothesis that reactive oxygens induce deletion mutations in mammalian cells.

The automation of routine calculations and graphing for alkaline filter elution analysis on a computer-controlled liquid scintillation analyzer.

The data calculations and graphing functions for the widely used alkaline filter elution technique have been completely automated. This saves considerable time and increases both efficiency and accuracy by eliminating human error. The automation was accomplished utilizing Lotus 1-2-3 and Lotus Graphwriter II on a Packard Tri-Carb 1900CA liquid scintillation analyzer containing a built-in pico-XTE computer. A batch file is used to control the overall execution of the process. It copies the data stored by the 1900CA into the Lotus subdirectory and invokes Lotus 1-2-3. A Lotus macro automatically imports the data file, performs the calculations, and prints the results. Graphwriter II is then invoked by the batch file and the charts are composed and graphed. Finally, the instrument operating software for the 1900CA is reentered and sample analysis can be resumed for any unanalyzed samples.

Mitomycin-induced chromatid breaks in HeLa cells: a consequence of incomplete DNA replication.

The formation of chromosome aberrations induced by alkylating agents such as mitomycin C has been shown to require the passage of the treated cell through S phase. However, the exact mechanisms by which mitomycin C-induced DNA lesions are translated into chromosome aberrations during S phase are not known. The purpose of these studies was to better understand the molecular basis of chromosome aberration formation after mitomycin C treatment. The morphology of metaphases of cells treated in G1 phase with mitomycin C resembled that of prematurely condensed chromosomes of S-phase cells. Consequently we postulated that chromosome aberrations resulted from cells reaching mitosis without completing DNA replication. This was tested by treating HeLa cells in G1 phase with mitomycin C and then analyzing these cells at mitosis for residual DNA damage and DNA content. Utilizing the DNA alkaline elution assay for DNA damage, we showed that HeLa cells progress through S phase into mitosis with intact DNA-DNA interstrand cross-links. These cross-links, originally induced into parental DNA, were associated equally with parental and newly replicated DNA at the time the cells reached mitosis. This suggests that recombinational events had taken place during the DNA replication process. Cells that were treated in G1 phase and allowed to proceed to mitosis in the presence of bromodeoxyuridine to density label newly replicated DNA were analyzed with cesium chloride density sedimentation. Unreplicated DNA was present in the mitotic cells of the treated populations but not in the untreated control cells. Further, flow cytometric measurements, made under hypotonic conditions in order to reduce chromatin condensation effects, demonstrated that the mitotic cells from the mitomycin C-treated populations contained 10-20% less DNA than untreated mitotic controls. These results indicate that chromosome breaks induced by mitomycin C are the result of cells reaching mitosis without having fully completed DNA replication.

Measurement of low levels of x-ray mutagenesis in relation to human disease.

We previously demonstrated that conventional methods for measurement of mutagenesis in mammalian cells are subject to serious error that causes underestimation of environmental contributions to cancer and genetic disease. This error has been corrected by use of somatic cell hybrids containing a single human chromosome on which the marker genes are carried and by using doses of mutagenic agents so low that little cell killing occurs. This method permits direct measurement of the effects of low doses of radiation and other mutagens without resort to the controversial extrapolation procedure customarily used to estimate effects of doses in the neighborhood of actual human exposures. The new data demonstrate that the true mutagenesis efficiency at the low doses of ionizing radiation that approximate human exposures is more than 200 times greater than those obtained with conventional methods. This methodology also permits evaluation of localized mutations, large and small chromosomal deletions, and nondisjunctional processes and can be used for mutagens that need metabolic activation as well as for cooperatively acting agents. The two opposing classical views that in mammalian cells extrapolation to low doses of x-radiation is linear, on the one hand, or involves a threshold, on the other, are both demonstrated to be incorrect at least for the conditions here considered. The actual curve exhibits a downward concavity so that the mutational efficiency is maximal at low doses. These data may have important implications for human health.

Loss of repairability of DNA interstrand crosslinks in Fanconi's anemia cells with culture age.

Some published reports have suggested a possible repair deficiency for DNA interstrand crosslinks in cells derived from patients with Fanconi's anemia (F.A.), that others, using different F.A. cell lines, were unable to confirm. A reinvestigation of the cell lines used in the original report might resolve this controversy. The purpose of this study, then, was to compare 2 F.A. fibroblast cell lines, FA9 and FA18 (previously reported to be repair-deficient), with a normal human line, Detroit 550, in their ability to repair nitrogen mustard (NM)- and mitomycin C (MMC)-induced crosslinks. The alkaline elution technique was used in the analysis of repairability. Prelabeled cells in quiescent phase were treated with NM or MMC for 1 h and crosslinks were assayed immediately after treatment and at 24 h after drug removal. Early passage F.A. cells repaired crosslinks to the same extent as normal, early passage cells. However, with increasing passage number, the F.A. cells demonstrated a corresponding decrease in their ability to repair NM-induced crosslinks. In contrast, the normal cells did not show any age-related decrease in their ability to repair NM-induced crosslinks. Approximately equivalent repair rates were observed in quiescent 550 and F.A. fibroblasts after MMC treatment. Exponential and quiescent Detroit 550 cell populations showed no difference in the repair rate of MMC-induced crosslinks. These results indicate that F.A. cells can repair crosslinks early in cell culture but this ability is nearly eliminated with increasing passage.

The relationship between DNA and chromosome damage after bleomycin treatment: dose-response measurements.

The molecular basis for chromosome aberration formation has been studied using the sensitive techniques of premature chromosome condensation and DNA alkaline elution. The dose response of Chinese hamster ovary cells to bleomycin treatment at the DNA and chromosome levels was compared. Each DNA elution curve showed a 2-component profile, with a more sensitive component apparent at low doses. The chromosome aberration curves also exhibited a 2-component profile when determined in G2-PCC; however, this phenomenon was less apparent when chromosome damage was enumerated in mitotic figures. These results suggest that differential sensitivity to bleomycin exists within the cellular chromatin. The effect of dose rate on aberration formation was examined by administering bleomycin at 2 concentrations that, with different treatment times, yielded equivalent amounts of DNA damage. The chromatid exchange rate was independent of dose rate, suggesting that rapidly repaired DNA lesions are not involved in the formation of exchanges.

The repair of bleomycin-induced DNA damage and its relationship to chromosome aberration repair.

Previous studies using the technique of premature chromosome condensation indicated that nearly one-half of the bleomycin-induced chromatid breaks and gaps in CHO cells could be repaired within 1 h (repair starting at 30 min) after treatment. Cycloheximide and streptovitacin A (but not hydroxyurea or hycanthone) inhibited chromosome repair. The purpose of this study was to measure the kinetics of DNA repair after bleomycin treatment using the alkaline elution technique and to determine whether various inhibitors could block this repair. After bleomycin treatment, the major proportion of the repair of DNA damage occurred within 15 min, with significant repair evident by 2 min. This fast repair component was inhibited by 0.2% EDTA. A slower repair component was observed to occur up to 60 min after bleomycin treatment. None of the inhibitors tested were found to have a significant effect on the repair of bleomycin damage at the DNA level. Since chromosome breaks were observed not to begin repair until after 30 min while over 50% of the DNA was repaired by 15 min, these results suggest that the DNA lesions that are repaired quickly are not important in the formation of chromosome aberrations. Further, since cycloheximide and streptovitacin A blocked chromosome repair but had little measurable effect on DNA repair, these results suggest that the DNA lesions responsible for chromosome damage represent only a small proportion of the total DNA lesions produced by bleomycin.