Venu Gopal

c-MYC and c-MYB expression in acute myelogenous leukemia

Monoclonal antibodies and flow cytometry were used to detect the expression of c-myc and c-myb in the bone marrow (BM) and peripheral blood (PB) cells of patients with acute myelogenous leukemia (AML). The expression of neither gene was correlated with the percent blast cells in the BM or PB nor was there a correlation between c-myc and c-myb expression. A wide range of expression of each gene was found within each FAB type of AML. Patients who had a high proportion of leukemia cells expressing c-myb were less likely to respond to remission induction therapy than patients in whom a low proportion of cells expressed c-myb. This association appears to reflect an inverse relationship between the proportion of cells expressing c-myb and the sensitivity of leukemia cells to the killing effects of chemotherapy in vivo. Treatment outcome was unrelated to c-myc expression.

Abnormal regulation of the myc gene in myeloid leukemia

To study the regulation of expression of the myc protooncogene, cells from normal individuals and patients with acute myelogenous leukemia (AML), and chronic phase and blastic crisis of chronic myeloid leukemia (CML) cells were put in overnight culture in the presence or absence of fetal calf serum. Myc expression in normal marrow cells and chronic phase CML cells fell after culturein vitro. In contrast, myc expression was maintained or increased in a majority of the AML and blastic crisis CML specimens. These data demonstrate that the regulation of myc expression is disordered in many AML and blastic crisis specimens but not in chronic phase CML cells.

Distribution of cell cycle times amongst the leukemia cells within individual patients with acute myelogenous leukemia

Seventeen patients with AML received infusions of BrdUrd to permit measurement of the cell parameters of leukemia cells in vivo. The range of S-phase times was measured by using a two color BrdUrd/Pl analysis to determine the range of BrdUrd incorporation into cells which had been in S-phase throughout the entire duration of BrdUrd administration. These data were, in turn, used to calculate the range of cell cycle times amongst the leukemia cells present within individual patients. The range of cell cycle times amongst the leukemia cells present within individual patients differs between patients, with some leukemia cell populations characterized by narrow and others by broad ranges. In general, the longer the mean cell cycle time (between 27 and 112 h) the broader the range of cell cycle times. These differences may help to explain, in part, the differences in response to therapy among patients whose mean leukemia cell kinetic parameters are similar.

Clonogenic potential of myeloid leukaemia cells in vitro is restricted to leukaemia cells expressing the CD34 antigen

Cells from patients with acute myeloid leukaemia (AML) or chronic myeloid leukaemia (CML) were separated into CD34-enriched and CD34-depleted subpopulations. The clonogenic capacities of these two subpopulations were then compared to each other and to the original unseparated cell population. In every study, the CD34-enriched subpopulation demonstrated a substantial increase in clonogenicity in vitro in comparison with the original cell population, while the reverse was the case for the CD34-depleted subpopulations. For reasons not clear at present, the enrichment for clonogenic cells far exceeded the enrichment for cells expressing the CD34 antigen. Additionally, the clonogenic potential was found to be unrelated to the level of myc expression in the various cell populations.

Comparison of Two Methods for Concentrating CD34 + Cells from Patients with Acute Non-Lymphocytic Leukemia

The aim of the present study was to compare two different methods for obtaining CD34+ cells from the peripheral blood or the bone marrow of patients with acute non-lymphocytic leukemia (ANLL). Twenty-two samples, obtained from 19 patients, were density cut (Ficoll-Hypaque 1.077) and, after incubation with My10 antibody, separated by panning or by immunomagnetic beads. Immunomagnetic beads provided a significantly better separation than panning, either in terms of concentration of CD34+ cells (85.5 +/- 11.6% vs. 55.7 +/- 25.0%, p = 0.003) or in terms of depletion of CD34+ cells (3.9 +/- 8.0% vs. 30.9 +/- 26.3%, p = 0.008). This was consistent with the virtually complete depletion of colony forming cells (CFC) in the CD34 negative fraction and the recovery of virtually all the CFC in the positive fraction in the samples separated by immunomagnetic beads. In conclusion, separation by immunomagnetic beads can allow collection of nearly pure CD34+ and CD34- cell populations from patients with ANLL, thereby facilitating the study of the biological characteristics of these cell populations. Moreover the method is less time consuming than panning and is not toxic to the CFC.

Effects of rhGM-CSF on Myeloid Clonogenic Cells in Acute Myelogenous Leukemia Patients

The effects of rhGM-CSF in vivo on the myeloid clonogenic cells present in 6 AML patients was evaluated. The relative number of clonogenic cells fell in 4 of the 6 patients. The effects of rhGM-CSF on the percentage of clonogenic cells in S phase and the sensitivity of clonogenic cells to cytosine arabinoside varied among the patients. These effects were not related to the effects of rhGM-CSF on the white blood cell count or on the proliferative rate of the leukemia cell population as a whole.

The multiple drug resistance gene, MDR1: Expression at the protein and RNA levels

A comparison of three different approaches to detect MDR1 expression in myeloid leukemia cells was undertaken. With respect to the 4 different antibodies studied, a high proportion of false positive reactions were detected. Substantial discordance between MDR1 expression as indicated by Northern blot analysis, PCR, and immunohistochemistry was found. These findings complicate the clinical interpretation of data derived from these methods.