The myb proto-oncogenes are thought to have a role in the cell division cycle. We have examined this possibility by genetic analysis in Drosophila melanogaster, which possesses a single myb gene. We have described previously two temperature-sensitive, recessive lethal mutants in Drosophila myb (Dm myb). The phenotypes of these mutants revealed a requirement for myb in diverse cellular lineages throughout the course of Drosophila development. We now report a cellular explanation for these findings by showing that Dm myb is required for both mitosis and prevention of endoreduplication in wing cells. Myb apparently acts at or near the time of the G 2 /M transition. The two mutant alleles of Dm myb produce the same cellular phenotype, although the responsible mutations are located in different functional domains of the gene product. The mutant phenotype can be partially suppressed by ectopic expression of either cdc2 or string, two genes that are known to promote the transition from G 2 to M. We conclude that Dm myb is required for completion of cell division and may serve two independent functions: promotion of mitosis, on the one hand, and prevention of endoreduplication when cells are arrested in G 2 , on the other.

Primary murine fetal hemopoietic cells were transformed with a fusion protein consisting of the ligand-binding domain of the estrogen receptor and a carboxylterminally truncated c-Myb protein (ERMYB). The ERMYB-transformed hemopoietic cells exhibit an immature myeloid phenotype when grown in the presence of b-estradiol. Upon removal of b-estradiol, the ERMYB cells display increased adherence, decreased clonogenicity and di erentiate to cells exhibiting granulocyte or macrophage morphology. The expression of the c-myc, ckit, cdc2 and bcl-2 genes, which are putatively regulated by Myb, was investigated in ERMYB cells grown in the presence or absence of b-estradiol. Neither c-myc nor cdc2 expression was down-regulated after removal of bestradiol demonstrating that di erentiation is not a consequence of decreased transactivation of these genes by ERMYB. While bcl-2 expression was reduced by 50% in ERMYB cells grown in the absence of bestradiol, there was no increase in DNA laddering, suggesting that Myb was not protecting ERMYB cells from apoptosis. In contrast, a substantial (200-fold) decrease in c-kit mRNA level was observed following di erentiation of ERMYB cells, and c-kit mRNA could be partially re-induced by the re-addition of b-estradiol. Furthermore, a reporter construct containing the c-kit promoter was activated when cotransfected with a Myb expression vector, providing further evidence of a role for Myb in the regulation of c-kit.

Chromosomal translocations involving the genes encoding the alpha and beta subunits of the Pebp2͞Cbf transcription factor have been associated with human acute myeloid leukemia and the preleukemic condition, myelodysplasia. Inv(16)(p13;q22) fuses the gene encoding the beta subunit of Pebp2 to the MYH11 gene encoding a smooth muscle myosin heavy chain (Smmhc). To examine the effect of the inv(16)(p13;q22) on myelopoiesis, we used the hMRP8 promoter element to generate transgenic mice expressing the Pebp2␤Smmhc chimeric fusion protein in myeloid cells. Neutrophil maturation was impaired in PEBP2␤MYH11 transgenic mice. Although the transgenic mice had normal numbers of circulating neutrophils, their bone marrow contained increased numbers of immature neutrophilic cells, which exhibited abnormal characteristics. In addition, PEBP2␤MYH11 inhibited neutrophilic differentiation in colonies derived from hematopoietic progenitors. Coexpression of both PEBP2␤MYH11 and activated NRAS induced a more severe phenotype characterized by abnormal nuclear morphology indicative of granulocytic dysplasia. These results show that PEBP2␤MYH11 can impair neutrophil development and provide evidence that alterations of Pebp2 can contribute to the genesis of myelodysplasia.

To study intracellular pathways by which the human papillomavirus 16 oncogene E7 participates in carcinogenesis, we expressed an inducible chimera of E7 by fusion to the hormone-binding domain of the estrogen receptor. The chimeric protein (E7ER) transformed rodent fibroblast cell lines and induced DNA synthesis on addition of estradiol. In coimmunoprecipitation experiments, E7ER preferentially bound p130 when compared to p107 and pRb. After estradiol addition, E7ER localization changed to a more intense intranuclear staining. Induction of E7 function was not correlated with binding to p130 or pRb but rather with intranuclear localization and modest induction of binding to p107.Human papillomavirus (HPV) infection of genital tract epithelium is associated with dysplastic changes in epithelial growth and is an important prerequisite for the development of cervical cancer. Two viral genes, E6 and E7, mediate transformation by HPV (1-4). The majority of cervical carcinomas contain truncated forms of HPV, which retains E6 and E7, integrated into the cellular genome (5-9). HPV16 E7 induces growth of cells in soft agar (1), stimulates DNA synthesis (10, 11), cooperates with ras to transform primary cells (12, 13), and overcomes G 1 arrest induced by serum deprivation, actinomycin D, or overexpression of p21 (14-17). The protein encoded by E7 has a region of shared sequence homology with both adenovirus E1A and SV40 large T antigen, which mediates binding to the retinoblastoma gene product pRb (13,18). By binding to pRb, E7 alters the interaction of pRb with the transcription factor E2F-1, resulting in activation of E2F-responsive genes (19). E7 protein from high-risk HPV types, HPV16 and HPV18, binds with a higher affinity to pRb than E7 protein from low-risk HPV types such as HPV6 and HPV11 (20). Therefore, the prevailing model is that E7 protein, by binding to pRb, promotes progression through the cell cycle. The role of E7 binding to other Rb-family members such as p107 and p130 has not been entirely elucidated. However, it is known that induction of B-myb transcription by E2F-1 is the result of E7 binding to p107 rather than to pRb (21).To assess the relationship between transformation and E7 binding to Rb-family members, we have constructed an inducible chimeric molecule consisting of HPV16 E7 fused in-frame to the hormone-binding domain of the estrogen receptor (ER), an approach used to render other oncogenes steroiddependent (22)(23)(24)(25)(26)(27). By using this inducible chimera of E7, we demonstrate that HPV16 E7 activity is correlated with intranuclear localization of the chimera. In addition, E7 binds to greater proportions of intracellular p130 and p107 than pRb in quiescent cells.