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Biomedical subjects

W T Bellamy

Publications and source records attributed to W T Bellamy.

24 records · Page 2Linked to original sources
PubMed1989

Role of glutathione and its associated enzymes in multidrug-resistant human myeloma cells.

Multidrug resistance (MDR) is a phenomenon associated with the emergence of simultaneous cross-resistance to the cytotoxic action of a wide variety of structurally and functionally unrelated antineoplastic agents. The present study was undertaken to determine if 8226 human myeloma cells possessing the MDR phenotype had an increased ability to resist the intercalating drug doxorubicin (DOX) via glutathione-based detoxification systems. Glutathione S-transferase (GST) was isolated by affinity chromatography, and the enzyme activity was assessed using 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates. There was no difference in overall GST activity between the sensitive and resistant cells. Using a cDNA probe (pGTSS1-2) for the human placental, anionic GST isoenzyme, no overexpression of mRNA for this isoenzyme was noted in the resistant line. When glutathione peroxidase activity (GSH-px) was assessed using either H2O2 or cumene hydroperoxide as substrate, again there was no difference in enzyme activity. Non-protein sulfhydryl (NPSH) levels were found to be elevated significantly in the resistant 8226/DOX40 subline (19.2 +/- 0.1 nmol NPSH/10(6) cells) as compared to the drug-sensitive parental subline 8226/S (11.6 +/- 1.9 nmol NPSH/10(6) cells) (P less than 0.001). In addition, when the 8226/DOX40 cells were cultured in medium without doxorubicin, there was a consistent decline in NPSH values reaching a steady state identical to that of the 8226/S cells. However, the decrease in NPSH level was not accompanied by a change in the level of doxorubicin resistance as assessed by colony-forming assays. Depletion of glutathione by D,L-buthionine-S,R-sulfoximine had no effect on doxorubicin sensitivity in either subline. Thus, it appears that GSH-based detoxification systems are not causally involved in maintaining the MDR phenotype in 8226 human myeloma cells; rather they appear to comprise an epiphenomenon associated with the resistance selection procedure.

Buthionine Sulfoximine↗
PubMed1988

Direct relation of DNA lesions in multidrug-resistant human myeloma cells to intracellular doxorubicin concentration.

Using a human myeloma cell line selected for resistance to doxorubicin (8226/DOX), which expresses the multidrug resistance phenotype, we studied the effects of drug accumulation on DNA damage and cytotoxicity in multidrug-resistant cells. The resistant 8226 subline showed a decrease in [14C]doxorubicin accumulation as compared to the sensitive (8226/S) subline at all time points investigated. DNA alkaline elution techniques were used to examine the formation of single and double strand breaks and DNA-protein cross-links following exposure to doxorubicin in both sensitive and resistant sublines. When 8226/S and 8226/DOX40 cells were exposed to the same extracellular concentration of doxorubicin there was a difference in the quantity of DNA lesions occurring, with the 8226/DOX40 line exhibiting less DNA damage. However, when the extracellular concentration of drug was adjusted to yield equivalent intracellular concentrations these differences were removed and both lines exhibited the same degree of damage for all three DNA lesions. The same DNA lesions were also studied in isolated nuclei from sensitive and resistant cells. Such a model removes any confounding factors due to drug accumulation such as altered cytosolic distribution and/or metabolism of drug. We observed no difference in the formation of single or double strand breaks, or DNA-protein cross-links when the nuclei were exposed to the same concentration of doxorubicin. Results from colony-forming assays have shown that when resistant and sensitive 8226 cells were exposed to high concentrations of doxorubicin, there was a good correlation between DNA damage, drug accumulation, and cytotoxicity. This relationship did not hold for lower concentrations of doxorubicin, for which there was a lack of correlation between drug accumulation and cytotoxicity. Such findings may possibly be due to a prolonged retention of drug by the sensitive cell line as compared to the resistant cells. Further studies are under way to examine this possibility.

Cell Survival↗
PubMed1988

Verapamil reversal of doxorubicin resistance in multidrug-resistant human myeloma cells and association with drug accumulation and DNA damage.

Verapamil reversed resistance to doxorubicin in a human multiple myeloma cell line selected for multiple drug resistance. The drug-resistant cell line 8226/DOX40 is known to have reduced intracellular drug accumulation associated with the overexpression of P-glycoprotein when compared to the sensitive parent cell line 8226/S. Verapamil alone was minimally cytotoxic in both cell lines, but reversed doxorubicin resistance in a dose-related manner in 8226/DOX40. A similar dose-response relationship was observed for verapamil in increasing net intracellular doxorubicin accumulation. This increased net accumulation was secondary to block of enhanced doxorubicin efflux by verapamil from resistant cells. In contrast, verapamil did not alter initial doxorubicin accumulation over the first 60 s when incubated with resistant cells. Addition of verapamil to the 8226/DOX40 cells enhanced the formation of doxorubicin-induced DNA single strand breaks, double strand breaks, and DNA-protein cross-links. Verapamil had no effect on these lesions in the drug-sensitive cells. In addition, verapamil did not affect chemotherapeutic cytotoxicity or transport in the drug-sensitive cell line. Verapamil appears to reverse doxorubicin resistance in this human myeloma cell line by blocking enhanced drug efflux, leading to increased drug accumulation and enhanced DNA damage.

Cell Survival↗
PubMed1988

Daily variation in non-protein sulfhydryl levels of human bone marrow.

There is increasing evidence that the dosing of certain antineoplastic agents based on a circadian type of schedule may have a beneficial effect on the outcome of patient therapy. Such regimens allow for a more intensive course of drugs to be administered due to a reduction in the toxicities associated with these agents. Since many of the antineoplastics in use today either form reactive intermediates or generate toxic free radical species within the cell, cellular thiols such as glutathione may play a role in their detoxification. Our studies were designed to investigate whether there exists a consistent daily fluctuation in the thiol content in human bone marrow samples. Five normal male volunteers, ages 26-32 years, underwent repeat bone marrow aspirations at approx. 8 a.m. and 8 p.m. Mean peak non-protein sulfhydryl (NPSH) levels of 47.7 nmole/mg protein occurred in morning bone marrow aspirates while evening aspirates showed markedly reduced mean levels of 7.9 nmole/mg protein. Reduced glutathione was used as a standard in these assays which measured sulfhydryls spectrophotometrically using Ellman's reagent. Our findings may help to explain the observed reduction in myelotoxicity in chemotherapy designed to take advantage of human circadian rhythms. These results support the proposition that the administration of certain cytotoxic drugs to match peak levels of thiols in the marrow may facilitate more intensive and active chemotherapy regimens.

Adult↗
PubMed1983

Biocidal braided sutures.

Once sutures become contaminated it is difficult for local tissue defenses or antibiotics to eliminate the bacteria and prevent infection. Impregnation of sutures with antibiotics before implantation is one way to prevent bacterial seeding of the suture surface. In this study, braided silk and Dacron sutures were impregnated with the antibiotic complex, neomycin palmitate. Using our standard wound model in the mouse, the efficacy of these biocidal sutures was determined in the presence of Staphylococcus aureus, Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa. Implantation of these biocidal sutures into tissue contaminated with 10(7) organisms resulted in substantially decreased numbers of bacteria as compared with that of tissue receiving control sutures. In most cases, the tissue bacterial counts in the presence of biocidal sutures were also considerably lower than that for similarly contaminated tissue without a suture. Consequently, the presence of the neomycin palmitate eliminated the infection-potentiating effect of the suture. The therapeutic benefit of the biocidal sutures was related to the dose of antibiotic complex and was not adversely affected by delaying suture implantation.

Animals↗