Biomedical subjects

D D Ross

Publications and source records attributed to D D Ross.

68 records · Page 4Linked to original sources

PubMed1984

Schedule-dependent enhancement of 1-beta-D-arabinofuranosylcytosine incorporation into HL-60 DNA by deoxyguanosine.

We studied the ability of the purine deoxynucleoside deoxyguanosine (dGuo) to enhance 1-beta-D-arabinofuranosylcytosine (ara-C) incorporation into DNA of HL-60 cultured human leukemia cells. The effects of dGuo on ara-C incorporation into DNA were compared to those of thymidine (dThd), a pyrimidine deoxynucleoside known to augment ara-C effects both in vitro and in vivo. Both deoxynucleosides doubled the cells in the S phase of the cell cycle within the exposure periods (up to 48 hr) and concentrations (10 to 1000 microM) tested. Both deoxynucleosides enhanced ara-C incorporation into DNA equally. However, dThd and dGuo differed in the schedule required to achieve this effect. Simultaneous exposure of cells to ara-C and dThd increased ara-C incorporation into DNA approximately 3.5-fold. Preincubation of cells with dThd for 16 hr prior to the addition of ara-C further enhanced ara-C incorporation into DNA (to approximately 5-fold) in direct proportion to the dThd-induced increase in cells in S phase. Preincubation was essential for dGuo, since 16-hr preincubation of cells with concentrations as low as 30 microM caused augmentation of ara-C incorporation into DNA; but simultaneous exposure of cells to dGuo and ara-C caused no augmentation of ara-C incorporation into DNA. The augmentation of ara-C incorporation into DNA caused by preincubation of the cells with dGuo results from a number of factors, including the cytokinetic effect of increasing the percentage of cells in S phase and the reduction of intracellular dCTP pools. Maximal dGuo enhancement of ara-C incorporation into DNA (approximately 5-fold) required greater than 100 microM dGuo, 16-hr preincubation with dGuo, and final incubation of cells with ara-C after removal of dGuo. We explain this further augmentation of ara-C incorporation into DNA caused by the removal of dGuo prior to adding ara-C by our observed inhibition of ara-C phosphorylation by dGuo concentrations greater than 100 microM.

Cell Line↗

PubMed1983

Persistence of herpes simplex virus type-2 genome in a human leukemic cell line.

To study the nature of virus-cell interaction in persistently infected cells we have examined production of infectious virus, synthesis of viral DNA and DNA polymerase in a human leukemic cell line K562. It was found that only one of three K562 cell lines was permissive for limited growth of HSV-2 and infectious virus was released in a cyclical fashion. Intranuclear inclusions with electron-dense fibrils and particles resembling viral structures were observed in the virus-infected but not control K562 cells. Viral DNA synthesis could not be detected by centrifugation in CsCl density gradients; but was readily identified by Southern blot hydridization of virus-infected intracellular DNA with purified viral DNA. Viral DNa polymerase was synthesized by infected cells during active infectious virus production. In one of the two K562 cell lines that did not produce infectious virus, a few DNA fragments from infected cells were found to hybridize with purified viral DNA. These results suggest that variable lengths of HSV-2 genome can be harbored and propagated by different human leukemic K562 cells.

Cell Line↗

PubMed1983

Detection in human serum by radioimmunoassay of histidyl-proline diketopiperazine, a metabolite of thyrotropin-releasing hormone.

Cyclo(His-Pro) is believed to be a metabolite of TRH. A specific antiserum directed against cyclo(His-Pro) was used to detect immunoreactive material in human serum. Gel filtration column chromatography was used to establish that cyclo(His-Pro)-like immunoreactivity was found not only in free form established to be identical to cyclo(His-Pro) by high pressure liquid chromatography analysis on reverse phase and cation exchange columns, but also in a fraction of about 100,000 mol wt. Free cyclo(His-Pro) was released by heating column fractions of 70,000 mol wt that were not immunoreactive before heating. A procedure for estimation of the content of free cyclo(His-Pro) in human serum was developed. Normal levels of cyclo(His-Pro) determined by this procedure were in the range of 11-33 pmol/ml. The level of cyclo(His-Pro) in sera of individuals with hypothyroidism, hyperthyroidism, or alcoholic cirrhosis was in the normal range, while patients with renal failure had approximately 3-fold elevated levels of the peptide. Analysis of human sera drawn at 3-h intervals over a 24-h period suggested a circadian rhythm of cyclo(His-Pro) levels.

Animals↗

PubMed1983

Variation amongst K562 cell cultures.

K562 cell cultures were obtained from three laboratories (A, B and C) outside our institution, and were designated according to source as K562A, K562B or K562C. The cultures obtained were constitutive or "wild type" K562 cell cultures, not cloned sublines. These cell cultures were compared with respect to growth kinetics, cell surface protein markers, surface antigens, cytogenetics and hemoglobin production. Morphology, growth kinetics in liquid suspension culture, cloning efficiency in soft agar culture, binding of anti-K562 monoclonal antibodies, and the majority of cell surface proteins were generally similar. In contrast, several important differences were observed: (1) hemoglobin synthesis induced by hemin was significantly different among K562A, B and C, K562A being most sensitive (P less than 0.05); (2) whereas more than 90% of K562A or C cells appeared to be Philadelphia chromosome (Ph1)-positive, less than 15% of K562B cells contained a Ph1; (3) membrane proteins (93 and 85 kilodalton) were identified in K562A, whereas only the 93 kilodalton protein was detected in K562B and neither of the proteins were detected in K562C. Our results indicate that K562 cells maintained in different laboratories can undergo tangible changes which may influence experimental results obtained in studies using these cells.

Autoradiography↗

PubMed1981

Growth inhibition by thymidine of leukemic HL-60 and normal human myeloid progenitor cells.

We compared the relative susceptibility of HL-60, a human acute progranulocytic leukemia cell line, and the normal human myeloid progenitor cell, the colony-forming unit in culture, to growth inhibition by thymidine. Normal human myeloid colony formation was more sensitive to thymidine than was HL-60 colony formation, the former being inhibited by greater than or equal to 0.5 mM thymidine and the latter by greater than or equal to 5.0 mM. Colony inhibition by thymidine was irreversible in both cases after a seven-day incubation of the colony-forming unit in culture in liquid medium enriched with thymidine and subsequent replating in thymidine-free soft agar. Thymidine-induced inhibition of HL-60 cloning could be partially prevented by deoxycytidine, whereas normal myeloid cloning could not, suggesting that high-concentration thymidine may affect processes necessary for cloning in addition to deoxyribonucleotide synthesis. HL-60 growth in liquid suspension culture could be suppressed transiently by 1.0 mM thymidine, suppressed totally by greater than or equal to 5.0 mM thymidine, and rescued completely by concomitant addition of deoxycytidine. The development of resistance to 1.0 mM thymidine could not be accounted for by reduction in thymidine pool size or by reduction in thymidine kinase activity. Replating of HL-60 cells growing in the presence of 1.0 mM thymidine in liquid medium in thymidine-free soft agar produced significant colony inhibition, also suggesting that thymidine affects more than just deoxyribonucleotide synthesis or that the clonogenic HL-60 cell presents a distinct subpopulation with more sensitive deoxyribonucleotide-synthetic control mechanisms.

Cell Differentiation↗

PubMed1981

Effects of deoxynucleosides on cultured human leukemia cell growth and deoxynucleotide pools.

We investigated the mechanism of cell growth inhibition caused by the deoxyribonucleosides thymidine (dThd), deoxyguanosine (dGuo), deoxyadenosine (dAdo), and deoxycytidine (dCyd). Growth of the cultured human leukemic cells HL-60 and K-562 was measured by cloning in soft agar. Of the deoxyribonucleosides, dGuo was the most potent cell growth inhibitor; however, the potency of added dAdo was probably attenuated by the presence of adenosine deaminase in the tissue culture growth medium. The concentrations of nucleoside causing 50% inhibition of HL-60 cloning were: dCyd, greater than 10,000 microM; dAdo, 500 microM; dThd, 5,000 microM; and dGuo, 80 microM. For K-562 cloning, the concentrations causing 50% inhibition of cloning were dCyd, greater 10,000 microM; dAdo, 1,600 microM; dThd, 880 microM;' and dGuo, 100 microM. Measurement of deoxycytidine 5'-triphosphate (dCTP) pool size in HL-60 cells following incubation with 750 microM deoxyribonucleosides revealed that dGuo caused the greatest reduction of dCTP pools, both in early (passage 10)- and late (passage 71)-passage-derived HL-60 cell cultures (35 and 19% of control, respectively), compared to dThd (61 and 26% of control, respectively) and dAdo (39% of control of HL-60 passage 10). In K-562 cells, reductions in dCTP pool size caused by dAdo, dThd, and dGuo were 68, 46, and 35% of control, respectively. Incorporation of [3H]dCyd into DNA of HL-60 and K-562 cells was enhanced by dThd and dGuo, but the degree of enhancement was greater for dThd than for dGuo. Despite its effect in reducing HL-60 dCTP pool size, dAdo failed to enhance [3H]dCyd incorporation in either HL-60 or K-562 cells. Addition of dCyd to the cultures could only partially rescue the inhibition of HL-60 cloning caused by dThd or dGuo, suggesting that inhibition of cytidine 5'-diphosphate reduction by ribonucleotide reductase is not the only mechanism whereby these nucleosides inhibit leukemic cell cloning. These data suggest that, in addition to inhibiting de novo dCTP production via ribonucleotide reductase, these nucleosides may affect other processes in the salvage pathway such as cellular uptake and phosphorylation or the DNA polymerase reaction itself.

Adenosine Deaminase↗

PubMed1980

Diurnal variation of circulating human myeloid progenitor cells.

We investigated whether human circulating myeloid progenitor cells (CFU(c)) exhibit diurnal variation. Peripheral venous blood samples were drawn every 3 h from each of the nine healthy male volunteers. Blood mononuclear cells were harvested by the Ficoll-Hypaque method. CFU(c) content of blood was determined by the soft agar cloning assay. Human placental conditioned medium (HPCM) was used as colony stimulating factor. We found a 24 h cyclic variation of colonies/10(5) mononuclear cells plated with a maximum at 9 a.m. (P < 0.01). Analysis of the colonies by in situ staining indicated that this variation could be attributed almost entirely to colonies composed of eosinophilic cells (representative of CFU(eos)). Neutrophil/macrophage colonies (representative of CFU(G,M)) exhibited a trend toward a 9 a.m. maximum, but this variation was not statistically significant. We conclude that time of blood collection should be standardized in experiments measuring human peripheral blood CFU(c).

Cells, Cultured↗

PubMed1979

Loss of terminal deoxynucleotidyl transferase (TdT) activity as a predictor of emergence of resistance to chemotherapy in a case of chronic myelogenous leukemia in blast crisis.

A 46-year-old female with chronic myelogenous leukemia (CML) in blast crisis was monitored for terminal deoxynucleotidyltransferase (TdT) activity of marrow and peripheral blood throughout the course of her illness. TdT was elevated at the time of diagnosis of blastic transformation, and the patient easily obtained remission after therapy with hydroxyurea, 6-mercaptopurine and prednisone. The patient enjoyed a remission of eight months duration, and at time of relapse, marrow TdT was again elevated. The patient again obtained complete remission with the same regimen, with the addition of vincristine, given weekly. This second remission was shortlived, however, and at relapse marrow TdT activity was undetectable. Subsequently, the patient failed to achieve remission, despite the use of a wide variety of chemotherapeutic agents. This case suggests that loss of TdT activity in blastic CML cells marks the emergence of cells resistant to existing chemotherapeutic agents.

Antineoplastic Agents↗

PubMed1979

Tamoxifen in advanced male breast cancer.

A 47-year-old man with metastatic carcinoma of the breast was found to have positive estrogen and progesterone receptors but refused castration or estrogens. After failing on combination chemotherapy he was started on tamoxifen, which resulted in a partial response lasting seven months. He relapsed in bone and then consented to orchiectomy, which resulted in further clinical improvement. Thus, the tamoxifen did not preclude further hormonal therapy. This case, added to others reported, suggests that tamoxifen may be useful in the treatment of advanced male breast cancer. Further studies are needed to determine the level of activity, when to use the drug, and the predictive value of hormone receptors.

Breast Neoplasms↗

PubMed1975

Effects of urinary proteins from certain leukemics upon macromolecular synthesis and enzyme levels in bone marrow cultures.

Urinary proteins from human leukemic patients have been found to alter quantitatively macromolecular synthesis in primary mouse bone marrow cultures. Urinary protein-stimulated incorporation of [3H]uridine into RNA was found after 1 day of culture. Increased levels of adenine phosphoribosyltransferase and lysozyme were demonstrable at 3 and 5 days, respectively, with urinary protein-supplemented cultures. The incorporation of 3H-labeled deoxynucleosides into DNA was higher in the presence of urinary proteins after 2 days of culture. The rate of incorporation of [3H]deoxyuridine into DNA was strongly inhibited by 10(-5) M Methotrexate and 10(-6) M 5-fluorodeoxyuridine, however, the effect of urinary proteins on incorporation of [3H]uridine into RNA and lysozyme accumulation were not inhibited. Urinary proteins also stimulated the formation of "colonies" (groups of at least 30 cells) in media containing methylcellulose. This latter phenomenon was also not inhibited by 10(-5) M Methotrexate or 10(-6) M 5-fluorodeoxyuridine. The results of these studies are consistent with the postulate that in the presence of human urinary proteins, mouse bone marrow cells in culture proceed to a phenotype characteristic of circulating peripheral white cells.

Adenine Phosphoribosyltransferase↗

PubMedDate not supplied

PBK/TOPK is a novel mitotic kinase which is upregulated in Burkitt's lymphoma and other highly proliferative malignant cells.

PBK/TOPK is a recently cloned serine/threonine kinase which is phosphorylated during mitosis. Earlier work indicated that this kinase is upregulated in a Burkitt's lymphoma cell line (GA-10). To determine whether PBK/TOPK is upregulated in other mitotically active neoplastic cell lines and tissues, Northern analysis was performed on a panel of malignant cell lines and on clinical samples from patients with leukemia or lymphoma. While PBK/TOPK mRNA was not detectable in normal peripheral blood cells and was weakly expressed in hyperplastic tonsillar B-cells, significantly higher levels of mRNA were detected in 8 Burkitt's lymphoma cell lines, 10 other neoplastic cell lines, and 2 clinical samples-one derived from a patient with ALL and a second derived from a patient with relapsed myeloma. In addition, Northern analysis of fetal tissues showed upregulated expression of PBK/TOPK in fetal kidney, lung, spleen, brain, and testis. These data suggest that PBK/TOPK expression is increased in highly proliferative malignant cells and during normal fetal development.

Adult↗

PubMedDate not supplied

Combinations of P-glycoprotein blockers, verapamil, PSC833, and cremophor act differently on the multidrug resistance associated protein (MRP) and on P-glycoprotein (Pgp).

Clinical studies are currently in progress to evaluate functional modifiers of P-glycoprotein (Pgp), an efflux pump associated with resistance to cancer chemotherapy. However, the effects of these modifiers on a more recently discovered efflux pump, the multidrug resistance associated protein (MRP), have not yet been fully characterized. MRP is expressed in most human tissues and is overexpressed in several tumor types. For these reasons, we have investigated the effects of three prototype Pgp modifiers, which act by different modes on the function of Pgp, on the function of MRP in two MRP-overexpressing cell lines: UMCC/VP lung and MCF-7/VP breast cancer cells. Clinically optimal plasma levels of verapamil, cremophor, and PSC833 have been shown to completely block the function of Pgp in Pgp-over expressing cells. However, in the two MRP-over expressing cell lines, these modifiers only partially blocked the function of MRP and combinations of these optimal concentrations acted antagonistically. Similar antagonistic effects were seen with combinations of suboptimal concentration levels of these blockers, while these combinations resulted in synergistic effects in Pgp overexpressing cells. For two biophysical parameters measured at the plasma membrane, membrane fluidity and membrane potential, the effects of these modifiers were essentially similar in Pgp and MRP expressing cells. We suggest that the 170 kD Pgp and the 190 kD MRP glycoproteins, imbedded in the plasma membranes, respond differently to simultaneous effects of the investigated prototype resistance modifiers. These results also suggest that the identification of the specific mechanism of drug resistance is important for the selection of chemotherapeutic strategies to block the efflux pump on the cancer cell.

3T3 Cells↗

PubMedDate not supplied

Deoxyguanosine enhancement of cytarabine nucleotide accumulation in human leukemia cells.

We studied the effect of deoxyguanosine (dGuo) on cellular cytarabine (ara-C) nucleotide accumulation of the human leukemia cell line K562 and of bone marrow blast cells derived from patients with acute nonlymphocytic leukemia. Exposure of cells in culture to dGuo increased ara-C nucleotide accumulation measured in cell lysate, with an average increase of 386% (range, 242%-537%) of control in the presence of 500 microM dGuo. Maximal elevation occurred after 8 hours of exposure and remained constant through 48 hours. dGuo also enhanced deoxycytidine nucleotide accumulation, but dGuo enhancement favored accumulation of ara-C nucleotides over dCyd nucleotides. In cell cycle kinetic studies using flow cytometry, dGuo slowed accumulation of cells with apparent S-phase DNA content in a concentration-dependent fashion. However, neither the rate nor the magnitude of this effect correlated with the increase in ara-C nucleotide accumulation. Since the increase in ara-C nucleotide accumulation caused by dGuo could be prevented by 5 micrograms/ml of cycloheximide, this process appears to require new protein synthesis. Although these data suggest that the elevation of ara-C nucleotide accumulation caused by dGuo may represent induction of enzyme synthesis, other possibilities are discussed. Exposure of bone marrow blast cells obtained from patients with acute nonlymphocytic leukemia to dGuo for 16 hours in liquid culture also increased ara-C nucleotide accumulation. In six of seven studies, exposure to dGuo in concentrations from 50 to 500 microM increased ara-C nucleotide accumulation from 160% to 3400%. These data suggest that dGuo may alter ara-C metabolism in a clinically useful fashion.

Arabinofuranosylcytosine Triphosphate↗

PubMedDate not supplied

Biochemical and clinical aspects of efflux pump related resistance to anti-cancer drugs.

This review paper will focus on the molecular biological, biochemical and biophysical aspects of the following three efflux pumps: P-glycoprotein (Pgp), Multidrug Resistance Protein (MRP) and Lung cancer Resistance-related Protein. Since suppression of the function of these pumps has clinical implications, novel approaches developed in our laboratory for blocking the function of Pgp and MRP are also discussed. The second part of this review will summarize the clinical significance and the results of clinical trials designed to suppress the effects of these efflux pumps.

ATP Binding Cassette Transporter, Subfamily B, Mem↗