Defense Date


Document Type


Degree Name

Doctor of Philosophy


Pharmacology & Toxicology

First Advisor

Eric H. Westin


Control of hematopoiesis is a complex set of events that is currently being dissected at the molecular level. To determine factors that may be crucial for commitment to terminal differentiation of myelomonocytic cells, a mutant of the HL-60 cell line was characterized at the cellular and molecular level. This clone, termed DMSOr, was shown to differentiate in a similar fashion as parental HL-60 in response to 1.3% DMSO at the morphologic and functional level. The anti-proliferative aspects of differentiation were also present in DMSOr as evidenced by decreased 3H-thymidine incorporation and an increased percentage of cells in the G0/G1 phase of the cell cycle. All of these phenotypic changes induced in DMSOr would revert if the DMSO was removed at any point during the differentiation process, thus DMSOr, despite its ability to functional differentiate, could not commit to terminal differentiation.

Associated with the altered phenotype of DMSOr was the altered expression of the proto-oncogene c-myb. Expression of cmyb remained detectable at 144 hrs of DMSO treatment in DMSOr but not HL-60. Similar findings were shown for the cell cycle other related genes. This altered gene expression did not extend to the c-myb related gene B-myb. The possibility that altered transcriptional regulation of c-myb was eliminated by nuclear run-on analysis and by the fact that a splice variant of c-myb with an altered 3' untranslated region showed no altered regulation. Thus the genetic defect in DMSOr may be in a global control factor for cell cycle related genes such as c-myb. This factor may regulate these genes at the post-transcriptional level.

To determine the mechanisms of regulation of c-myb during hematopoietic cell differentiation, transcriptional and posttranscriptional studies of c-myb following treatment of HL-60 cells with various differentiation inducers were undertaken. Retinoic acid and vitamin D3 regulated c-myb at the transcriptional level via an attenuator, while DMSO and phorbol dibutyrate activated multiple mechanisms of regulation. These included attenuation and a post-transcriptional regulation that was dependent on continuous transcription, but not translation in the case of DMSO. Phorbol ester regulation of c-myb occurred at the level of an attenuator and possibly a promoter at the transcriptional level. In addition there was post-transcriptional control of c-myb by phorbol dibutyrate that differed from the regulation by DMSO through the lack of transcriptional dependency. Thus c-myb is regulated at the transcriptional and the post-transcriptional level in an agent specific fashion during HL-60 differentiation.

A 2.4 kb message is present in the Northern blots probed for c-myb expression. This lower molecular weight message is regulated in an abberrant fashion compared to normal message during HL-60 differentiation. Probing of blots with different regions of a full length c-myb eDNA and primer extension analysis suggest that the 2.4 kb message may start in the exon of the c-myb locus.


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