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The ability of oncogenic proteins to regulate the rate of translation of specific mRNA subsets may be a rapid and efficient mechanism to modulate the levels and, in many cases, the activity of the corresponding proteins. In the past few years, we have identified several RNA binding proteins with translation regulatory activity whose expression is markedly activated in the blast crisis of chronic myelogenous leukemia, which represents the most malignant disease stage. Perturbation of the activity of some RNA binding proteins suppresses the leukemogenic potential of BCR/ABL-expressing cells. Most importantly, we have identified some of the targets of these RNA binding proteins. Two of these targets, c/ebp alpha and mdm2 mRNAs, are directly relevant for the altered differentiation and survival of leukemic cells. The identification of mRNA targets translationally regulated by RNA binding proteins overexpressed in tumor cells may lead to the development of therapeutic strategies aimed at modulating the translation rate of specific mRNAs.

Shuttling hnRNPs control the fate of eukaryotic mRNAs throughout their journey from the active site of transcription to that of translation; thus, gain or loss of their function in hematopoietic cells might result in altered hematopoiesis and/or be associated with the process of leukemogenesis. In BCR/ABL-expressing cells, there is a marked increase in the protein levels FUS, hnRNP A1 and hnRNP E2, three RNA-binding proteins involved in the regulation of mRNA processing, nucleocytoplasmic export, and translation. Ectopic expression and/or inhibition of the activity of these RNA-binding proteins affects proliferation, survival, and differentiation of normal and BCR/ABL-expressing cells, suggesting that enhanced expression/activity of certain RNA-binding proteins plays an important, but as yet unrecognized, role in BCR/ABL leukemogenesis. The identification of the mRNA subsets associated with RNA-binding proteins upregulated in BCR/ABL-expressing cells should functionally link the process of leukemogenesis with alteration of mRNA metabolism.

B-Myb is a transcription factor belonging to the myb family, whose activity has been associated with augmented DNA synthesis and cell cycle progression. We showed recently that B-Myb autoregulates its own expression through promoter transactivation. We report in this study that CDK9, the cyclin T associated kinase, which phosphorylates and activates RNA-Polymerase II, suppresses B-Myb autoregulation through direct interaction with the carboxyl-terminus of the B-Myb protein. Down-regulation of the transactivating ability of B-Myb is independent of the kinase activity of CDK9, because a kinase deficient mutant (dn-CDK9) also represses B-myb gene autoregulation. Overexpression of CDK9 did not result in suppression of p53-dependent transactivation or inhibition of the basal activity of the promoters tested so far, demonstrating that CDK9 is a B-Myb-specific repressor. Rather, transfection of the dominant negative dn-CDK9 construct inhibited the basal activity of the reporter genes, confirming an essential role for CDK9 in gene transcription. In addition, Cyclin T1 restores B-Myb transactivating activity when co-transfected along with CDK9, suggesting that the down-regulatory effect observed on B-Myb is specifically due to CDK9 alone. Thus, our data suggest that CDK9 is involved in the negative regulation of activated transcription mediated by certain transcription factors, such as B-Myb. This may indicate the existence of a feedback loop, mediated by the different activities of CDK9, which links basal with activated transcription.

Tk-ts13 cells are BHK-derived fibroblasts that have a G1-specific temperature sensitive (ts) mutation so that the cells arrest in the G1 phase of the cell cycle at the restrictive temperature of 39.6 degrees. We have introduced into these cells a plasmid carrying the human c-myb cDNA under the control of the early SV40 promoter. The resulting cell line, ts13 myb cells, express the c-myb RNA and protein and, when serum-stimulated, they undergo one round of DNA replication even at the restrictive temperature. Under the same conditions, the parent cell line (tk-ts13 cells) are blocked in G1 and fail to replicate DNA. We have investigated the expression of two late growth-regulated genes, PCNA and histone H3, in tk-ts13 and in ts 13 myb cells, both at permissive and restrictive temperature. At the permissive temperature of 34 degrees, the mRNA levels for PCNA and histone H3 increase markedly after serum stimulation in both cell lines, reaching a peak at the time of DNA synthesis. At the restrictive temperature of 39.6 degrees, the mRNA's for PCNA, and histone H3 are detectable in serum-stimulated ts13 myb cells while they are not detectable in the parent tk-ts13 cell line. Run-on transcription assays indicate that these 2 genes are transcribed in tk-ts13 cells equally well at permissive and nonpermissive temperatures, and that the presence of the myb product does not significantly increase their rates of transcription. The stability of the respective mRNA's is roughly the same at either temperature and in both types of cells. These results indicate: (1) a constitutively expressed c-myb gene product confers to fibroblasts the ability of temporarily by-passing a ts block in the G1 phase of the cell cycle and (2) the myb product, under these conditions, regulates the mRNA levels of PCNA and histone H3 either directly or indirectly by a post-transcriptional mechanism.