Stimulation of hematopoiesis in patients with bone marrow failure and in patients with malignancy by recombinant human granulocyte-macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotential hematopoietin. To assess the toxicity and biological activity of recombinant human GM-CSF (rhGM-CSF) in vivo, 25 patients with malignancy or bone marrow failure were treated with rhGM-CSF (specific activity approximately 5 x 10(7) U/mg) as part of a phase 1 trial. The treatment was administered by continuous intravenous (IV) infusion daily for 2 weeks at fixed dose levels and repeated after a 2- week rest period. Over the entire dose range tested (15 to 500 micrograms/m2/d), rhGM-CSF treatment was associated with dramatic increases (two- to 70-fold) in total leukocyte counts, which consisted predominantly of neutrophils, bands, eosinophils, and monocytes. Furthermore, six of the 14 patients with one or more cytopenias that received at least two cycles of treatment had multilineage responses characterized by twofold or greater increases in platelet count to a level above 100,000, twofold or greater increases in corrected reticulocyte count, and a reduced requirement for red cell transfusions. Three of these patients became independent of both red cell and platelet transfusions for 17 to 37 weeks of follow-up. Treatment was associated also with an increase in bone marrow cellularity and frequency of cycling progenitor cells. The treatment was well tolerated; side effects included constitutional symptoms and bone pain. These results demonstrated that rhGM-CSF has a significant impact on hematopoiesis in patients with advanced malignancy and also in patients with bone marrow failure.     

Premature chromosome condensation studies in human leukemia. I. Pretreatment characteristics.

The phenomenon of premature chromosome condensation (PCC) was used to compare the bone marrow proliferation characteristics of 163 patients with various forms of leukemia prior to the initiation of new therapy. The proliferative potential index (PPI, or fraction of G1 cells in late G1 phase) and the fraction of cells in S phase was determined and compared to the type of disease and the bone marrow blast infiltrate for each patient. Previously untreated patients with acute leukemia exhibited an average PPI value three times that of normal bone marrow (37.5% for acute myeloblastic leukemia [AML], acute monomyeloblastic leukemia [AMML], or acute promyelocytic leukemia [APML] and 42% for acute lymphocytic leukemia [ALL] or acute undifferentiated leukemia [AUL]). Untreated chronic myelogenous leukemia (CML) patients showed intermediate PPI values (25.2%), whereas CML patients with controlled disease exhibited nearly normal PPI values (14.6%). On the other hand, blastic-phase CML patients exhibited PPI values closer to that observed in patients with acute leukemia (35.4%). Seven patients with chronic lymphocytic leukemia (CLL) exhibited even higher PPI values. No correlations were observed between PPI values, fraction of cells in S phase, and marrow blast infiltrate. For untreated acute disease patients, PPI values were prognostic for response only at low and high PPI values. These results suggest that the PCC-determined proliferative potential is a biologic reflection of the degree of malignancy within the bone marrow.     

Model for the formation of double minutes from prematurely condensed chromosomes of replicating micronuclei in drug-treated Chinese hamster ovary cells undergoing DNA amplification

Double minutes (DM) have been associated with gene amplification in drug-resistant cells and tumor cells. However, the mechanisms by which DM are formed have not been elucidated. We present here a model to describe a possible mechanism of DM formation based on the observations made in two independent early drug-selected multidrug-resistant cell lines and from in vitro somatic cell fusion experiments between synchronized S- and M-phase cells. The multidrug-resistant cell lines contain both DM and amplified mdr (P-glycoprotein) gene. Cytogenetic analyses of cells at early stages of selection revealed the presence of a number of micronuclei in a subpopulation of these cells. These micronuclei were often asynchronous in their progression through the cell cycle. As a result, premature condensation of micronuclear chromatin was often observed in metaphase plates. The pulverized chromatin pattern seen in certain instances of S-phase prematurely condensed chromosomes displays a striking resemblance to DM structures. These DM-like structures are linked by replicating DNA as revealed by DNA labeling experiments. Somatic cell hybrids between S- and M-phase cells when grown in vitro demonstrated that S-phase prematurely condensed chromatin indeed gives rise to extra chromosomal structures in the successive cell generations. It is hypothesized that distinct DM-like structures may arise from the partially replicated and prematurely condensed S-phase chromosomes following their liberation as extra chromosomal entities after replication and/or recombination in the succeeding division cycle(s). The enrichment for DM containing specific genes in drug-resistant cells may result from the subsequent drug selections.     

Postnatal decline in pyridoxal phosphate and riboflavin. Accentuation by phototherapy

Riboflavin is a cofactor in the conversion of pyridoxine (vitamin B6) to pyridoxal phosphate (PALP), an essential coenzyme in numerous metabolic pathways, including neurotransmitter synthesis. Riboflavin and pyridoxine are light sensitive in vitro, and conflicting results have been reported on the in vivo effects of phototherapy on riboflavin. We studied 25 full-term neonates receiving phototherapy and 16 healthy controls to evaluate their riboflavin and PALP status. Both vitamin cofactors decreased in both sets of infants, but significantly more so in the irradiated group. While the biologic or clinical importance of a modest biochemical decline in the level of PALP has not been established, it is possible that transient behavioral changes in irradiated, jaundiced neonates could be mediated by decreased availability of PALP. The mechanism for the postnatal decline and the desirability of routine supplementation with pyridoxine, especially in irradiated infants, require further study.     

Differential expression of DNA topoisomerases I and II during the eukaryotic cell cycle.

We have utilized antibody probes to examine the expression of DNA topoisomerases I and II and chromosome scaffold protein Sc-2 in normal and transformed cells. Neither topoisomerase I nor Sc-2 shows significant fluctuations in content or stability across the cell cycle. In contrast, topoisomerase II undergoes significant cell cycle-dependent alterations in both amount and stability. As cells progress from mitosis into G1, much of the topoisomerase II is degraded. During the first 2 hr of G1, the half life of topoisomerase II is decreased from that measured in asynchronous cell populations by a factor of 7. This suggests that the chromosome condensation/decondensation cycle is coupled to a parallel cycle of synthesis and degradation of topoisomerase II. In control experiments, we also found that the half-life of topoisomerase II is shorter in normal cells than in transformed cells by a factor of 4. Since the number of copies of topoisomerase II per cell is also lower in normal cells, this suggests that control of topoisomerase II stability is altered upon transformation. The stability of topoisomerase I and Sc-2 does not differ significantly between normal and transformed cells.     

Simultaneous cell type identification and premature chromosome condensation analysis in a case of multiple myeloma

The technique of premature chromosome condensation was combined with immunocytochemical techniques to determine the karyotype of the plasma cells in a patient with multiple myeloma. Although the patient's myeloma cells had a diploid DNA content, the mean chromosome number was 39. Multiple chromosome rearrangements were documented in the G- and C-banded G1 and G2 prematurely condensed chromosomes, and several of these involved telomeric regions resulting in dicentric chromosomes. That the aberrant karyotype was present in the kappa light chain positive plasma cells was proved by simultaneous chromosome analysis and immunocytochemical examination of the fused cells. Thus the combination of premature chromosome condensation and immunocytochemistry proved a powerful tool for cytogenetic analysis of a slow-growing, heterogeneous cell population.     

Kinetics and extent of repair of bleomycin-induced chromosome damage in quiescent normal human fibroblasts and human mononuclear blood cells

The kinetics of chromosome damage repair after bleomycin treatment was studied in quiescent mononuclear human blood cells and human fibroblasts using the technique of premature chromosome condensation. Quiescent cells were treated with bleomycin for 30 min, washed free of drug, and fused with mitotic HeLa cells after various repair times. The amount of chromosome damage remaining was assayed in the G1 prematurely condensed chromosomes. The chromosome repair kinetics profile exhibited fast- and slow-repair components. The fast chromosome repair component was apparent within 2 hr after bleomycin treatment, with a significant amount of repair having occurred within 30 min. The absolute rate of chromosome repair then significantly slowed beyond 2 hr after treatment. The rate of chromosome repair was slightly dependent on dose with a higher rate observed at a higher dose. Interestingly, while quiescent fibroblasts were more sensitive to bleomycin than were mononuclear blood cells, both cell populations exhibited similar repair kinetics. These results suggest the following: (a) quiescent human fibroblasts and mononuclear blood cells show similar chromosome repair kinetics after bleomycin treatment; (b) there exist both fast- and slow-repair components after bleomycin treatment; and (c) the rate of chromosome repair is dependent on the degree of initial damage.