Adriamycin resistance in human tumor cells associated with marked alteration in the regulation of the hexose monophosphate shunt and its response to oxidant stress.

We found that Adriamycin increased the pentose phosphate shunt activity in both Adriamycin-sensitive (WT) and Adriamycin-resistant (ADRR) human breast cancer MCF-7 cells. In contrast, hydrogen peroxide and cumene hydroperoxide markedly stimulated pentose-shunt activity in ADRR but only moderately increased the activity in WT cells. Furthermore, the altered oxidation-reduction regulation is associated with changes intrinsic to the key enzymes of the pentose-shunt pathway, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase and with glutathione peroxidase. We found the Vmax values for glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were 50- and 4-fold lower, respectively, in ADRR than WT cells and the Kms of NADP+ were 10-fold lower in ADRR than WT. The activity of glutathione reductase in ADRR is 42% of that in WT. In spite of these changes, the response of the cells to both hydrogen peroxide and organic peroxide is not limited by either the capacity of the pentose shunt or glutathione reductase, but is determined by the activity of glutathione peroxidase and a glutathione transferase which possess peroxidase activity. The kinetic properties of the glucose-6-phosphate dehydrogenase in ADRR may, however, seriously limit the activity of cytochrome P-450 reductase, a major enzyme of Adriamycin conversion to a free radical.

The biology of tumor growth in the non-Hodgkin's lymphomas. A dual parameter flow cytometry study of 220 cases.

Dual parameter flow cytometry studies (cell DNA content and electronic cell volume) were performed in 220 cases of non-Hodgkin's lymphoma. All cases were characterized as B or T cell malignancies, based on immunologic surface marker characteristics. Aneuploidy by flow cytometry was more common among the B cell lymphomas than among the T cell lymphomas, and was most common among the large B cell lymphomas and B cell lymphomas of intermediate size. Ploidy index distributions showed a prominent hyperdiploid peak, as well as tumor cell populations with near-tetraploid DNA contents. In serial studies, a decrease in ploidy index was observed in association with clinical and histologic transformation in one case. The highest S fractions were observed among the large and intermediate B cell lymphomas and among the aggressive T cell lymphomas. In clinical samples consisting of mixtures of diploid and aneuploid populations, the data on the aneuploid components could often be separated from other components of the mixture in multiparameter studies on the basis of the larger electronic cell volumes of the aneuploid cells. In each case, the aneuploid large cell component almost invariably had a higher S fraction than the residual component(s) of the mixture. Overall, the data are consistent with a model of clonal selection and clonal evolution in the lymphomas in which early cytogenetic abnormalities that involve little or no change in total cell DNA content are followed by cell tetraploidization that is associated with cytogenetic instability and chromosome loss over the course of time.

Effects of vincristine on cell survival, cell cycle progression, and mitotic accumulation in asynchronously growing Sarcoma 180 cells.

The effects of vincristine (VCR) on cell survival, cell cycle progression, DNA synthesis, and metaphase accumulation were studied in relation to drug concentration and drug exposure duration in Sarcoma 180 cells in vitro. VCR was found to affect cells in interphase, producing a transient G2 block at all drug concentrations and drug exposure durations studied. VCR did not affect DNA synthesis directly. Increases in the metaphase index were delayed and always peaked at approximately 8 hr after drug removal, regardless of the duration of drug exposure. Increases in the metaphase index of sufficient magnitude to be commensurate with VCR lethality were observed only with prolonged drug exposure. VCR produced both nuclear fragmentation and polyploidy. The proportion of cells undergoing polyploidy increased progressively with increasing drug exposure duration. Interference with cytokinesis during prolonged VCR exposure may represent a lethal effect of VCR that is separate from its short-term effects. This could serve as the basis for the clinical study of the antitumor effects of prolonged VCR infusions.

Schedule dependence of vincristine lethality in Sarcoma 180 cells following partial synchronization with hydroxyurea.

The effects of exposure to 0.1, 0.5, or 2 microM vincristine for 4 hr were studied in Sarcoma 180 cells at various times after synchronization with 5 mM hydroxyurea for 1 hr. Maximum sensitivity to the lethal effects of vincristine was observed at 10 to 14 hr after hydroxyurea exposure at the higher vincristine concentrations, compared to a period of a maximum sensitivity to a second dose of hydroxyurea at 8 to 12 hr. Serial flow cytometry studies indicated that the apparent decrease in sensitivity to vincristine at 14 to 18 hr was due to the division of cells in the leading segment of the synchronized wave and their entry into the relatively resistant G1 phase prior to vincristine exposure. Synchronized cells that had not divided at the time of vincristine exposure were blocked transiently in G2. Serial metaphase index studies suggested that the G2 cells closest to the end of the cell cycle at the time of vincristine exposure were likely to exhibit the greatest degree of mitotic disorganization when they overcame the G2 block and entered metaphase. The present studies suggest that sensitivity to vincristine increases progressively as cells approach mitosis. The molecular mechanisms underlying this phenomenon are considered in relation to the increase in cell tubulin content during the course of cell cycle progression.

Automated radioautographic grain counting. Correction for grain overlap.

An algorithm is described for the calculation of radioautographic cell grain count from measurements of total cell nuclear area and total grain area. This algorithm provides a statistical correction for grain overlap that is based on the solution to the occupancy problem in probability theory. This method permits the use of automated grain counting over a wide range of grain counts per cell, and extends the useful dynamic range of radioautographic grain counting to well over 200 grains/cell.

Increased relative effectiveness of doxorubicin against slowly proliferating sarcoma 180 cells after prolonged drug exposure.

Doxorubicin (ADR) lethality was studied in Sarcoma 180 cells grown in vitro during log phase and early plateau phase. For both log phase and plateau phase cells, ADR lethality increased progressively with continuous drug exposure, and was dependent on drug concentration at every point in time. For any given level of toxicity to log phase cells, greater killing of plateau phase cells was achieved with prolonged drug exposure. The inhibition of DNA synthesis by ADR was delayed in onset, and increased gradually, both at low and high drug concentrations. The data suggest that the dependence of ADR lethality on proliferation rate and the higher therapeutic ratio in plateau phase cells with prolonged drug exposure are not directly related to the ADR effects on DNA synthesis.

Schedule-dependent synergism of combinations of hydroxyurea with adriamycin and 1-beta-D-arabinofuranosylcytosine with adriamycin.

The lethal effects of combinations of adriamycin (ADR) and either hydroxyurea (HU) or 1-beta-d-arabinofuranosylcytosine (ara-C) were studied in relation to drug-scheduling interval in Sarcoma 180 cells grown in vitro. Drug lethality was determined by cell cloning in soft agar. Serial kinetic changes induced by a priming dose of HU or ara-C were monitored by flow cytometry and related to schedule-dependent cell killing by ADR. All drug exposures were for 1 hr. When ADR was given together with either HU or ara-C, cell log kill was additive. However, when ADR was given after exposure to either HU or ara-C, cell killing was increased up to 200- to 500-fold, respectively. Maximum schedule-dependent synergism was observed at a drug-scheduling interval of 2 hr; schedule-dependent synergism decreased as the interval between drugs was increased beyond 2 hr. Schedule-dependent synergism was not observed when the same drug combinations were given in reversed order. Drug-induced changes in the DNA histogram were not seen until 5 hr after HU exposure and 8 hr after ara-C exposure. Thus, the schedule-dependent synergism between ADR and either HU or ara-C cannot be explained by cell cycle blockade with synchronization of cells in S phase.