Decreased retinoblastoma protein expression in acute myeloblastic leukaemia is associated with the autonomous proliferation of clonogenic blasts

Abstract

We have previously shown that blasts from some patients with acute myeloblastic leukaemia (AML) grow autonomously in vitro and that this growth pattern is related to the production of autocrine growth factors including granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-1 beta. However, another potential mechanism of autonomous growth involves the deletion of growth inhibitory molecules. One such inhibitory protein is the product of the retinoblastoma (Rb) gene which is expressed in normal haemopoietic cells and functions in cell cycle control and as a transcriptional repressor. Deletion of the Rb gene has been reported in various types of malignancy. We have examined the expression of the Rb gene product in blasts from 39 patients with AML by Western analysis. Using an antibody, Rb (Ab-2) which recognizes the C-terminal region, 11/39 samples (28%) failed to express Rb protein. The results of the Western blot analysis were confirmed by flow cytometry using a second antibody, Rb (Ab-1), against residues 300-380 of the Rb protein. Samples from seven of the 11 Rb-negative cases were studied for the expression of Rb mRNA; Rb mRNA was absent in four and three cases were positive. Analysis of the growth characteristics of these cells showed that blasts from 24/39 cases (62%) had partially or totally autonomous growth in a blast cell colony assay. Of these 24 samples with autonomous growth, 10 (41%) were Rb protein negative; in contrast, of the 15 cases with non-autocrine growth only one was Rb protein negative (7%, P < 0.05). Also, in Rb positive blasts, suppression of Rb protein production using an antisense oligonucleotide significantly increased proliferation of clonogenic AML blasts, confirming that the Rb protein acts as a negative regulator of growth in AML blasts. Our data suggest that deletion of Rb protein expression is frequently found in AML and is associated with the acquisition of autocrine growth characteristics, possibly as the consequence of derepression of genes involved in growth control and cytokine production.