The acute myeloid leukemia (AML)-associated CBF beta-SMMHC fusion protein impairs hematopoietic differentiation and predisposes to leukemic transformation. The mechanism of leukemia progression, however, is poorly understood. In this study, we report a conditional Cbfb-MYH11 knockin mouse model that develops AML with a median latency of 5 months. Cbf beta-SMMHC expression reduced the multilineage repopulation capacity of hematopoietic stem cells (HSCs) while maintaining their numbers under competitive conditions. The fusion protein induced abnormal myeloid progenitors (AMPs) with limited proliferative potential but leukemic predisposition similar to that of HSCs in transplanted mice. In addition, Cbf beta-SMMHC blocked megakaryocytic maturation at the CFU-Meg to megakaryocyte transition. These data show that a leukemia oncoprotein can inhibit differentiation and proliferation while not affecting the maintenance of long-term HSCs.

Acute myeloid leukemia subtype M4 with eosinophilia is associated with a chromosome 16 inversion that creates a fusion gene CBFB-MYH11. We have previously shown that CBFB-MYH11 is necessary but not sufficient for leukemogenesis. Here, we report the identification of genes that specifically cooperate with CBFB-MYH11 in leukemogenesis. Neonatal injection of Cbfb-MYH11 knock-in chimeric mice with retrovirus 4070A led to the development of acute myeloid leukemia in 2-5 months. Each leukemia sample contained one or a few viral insertions, suggesting that alteration of one gene could be sufficient to synergize with Cbfb-MYH11. The chromosomal position of 67 independent retroviral insertion sites (RISs) was determined, and 90% of the RISs mapped within 10 kb of a flanking gene. In total, 54 candidate genes were identified; six of them were common insertion sites (CISs). CIS genes included members of a zinc finger transcription factors family, Plag1 and Plagl2, with eight and two independent insertions, respectively. CIS genes also included Runx2, Myb, H2-T24, and D6Mm5e. Comparison of the remaining 48 genes with single insertion sites with known leukemia-associated RISs indicated that 18 coincide with known RISs. To our knowledge, this retroviral genetic screen is the first to identify genes that cooperate with a fusion gene important for human myeloid leukemia.

Polyploidy is often an early event during cervical carcinogenesis, and it predisposes cells to aneuploidy, which is thought to play a causal role in tumorigenesis. Cervical and anogenital cancers are induced by the high-risk types of human papillomavirus (HPV). The HPV E6 oncoprotein induces polyploidy in human keratinocytes, yet the mechanism is not known. It was believed that E6 induces polyploidy by abrogating the spindle checkpoint after mitotic stress. We have tested this hypothesis using human keratinocytes in which E6 expression induces a significant amount of polyploidy. We found that E6 expression does not affect the spindle checkpoint. Instead, we provide direct evidence that E6 is capable of abrogating the subsequent G 1 arrest after adaptation of the mitotic stress. E6 targets p53 for degradation, and previous studies have shown an important role for p53 in modulation of the G 1 arrest after mitotic stress. Importantly, we have discovered that E6 mutants defective in p53 degradation also induce polyploidy, although with lower efficiency. These results suggest that E6 is able to induce polyploidy via both p53-dependent and p53-independent mechanisms. Therefore, our studies highlight a novel function of HPV E6 that may contribute to HPV-induced carcinogenesis and improve our understanding of the onset of the disease. [Cancer Res 2007;67(6):2603-10]

Human papillomavirus (HPV) infection is necessary but not sufficient for cervical carcinogenesis. Genomic instability caused by HPV allows cells to acquire additional mutations required for malignant transformation. Genomic instability in the form of polyploidy has been demonstrated to play an important role in cervical carcinogenesis. We have recently found that HPV-16 E7 oncogene induces polyploidy in response to DNA damage; however, the mechanism is not known. Here we present evidence demonstrating that HPV-16 E7-expressing cells have an intact G 2 checkpoint. Upon DNA damage, HPV-16 E7-expressing cells arrest at the G 2 checkpoint and then undergo rereplication, a process of successive rounds of host DNA replication without entering mitosis. Interestingly, the DNA replication initiation factor Cdt1, whose uncontrolled expression induces rereplication in human cancer cells, is upregulated in E7-expressing cells. Moreover, downregulation of Cdt1 impairs the ability of E7 to induce rereplication. These results demonstrate an important role for Cdt1 in HPV E7-induced rereplication and shed light on mechanisms by which HPV induces genomic instability.

High-risk types of human papillomavirus (HPV) are considered the major causative agents of cervical carcinoma. The transforming ability of HPV resides in the E6 and E7 oncogenes, yet the pathway to transformation is not well understood. Cells expressing the oncogene E7 from high-risk HPVs have a high incidence of polyploidy, which has been shown to occur as an early event in cervical carcinogenesis and predisposes the cells to aneuploidy. The mechanism through which E7 contributes to polyploidy is not known. It has been hypothesized that E7 induces polyploidy in response to mitotic stress by abrogating the mitotic spindle assembly checkpoint. It was also proposed that E7 may stimulate rereplication to induce polyploidy. We have tested these hypotheses by using human epithelial cells in which E7 expression induces a significant amount of polyploidy. We find that E7-expressing cells undergo normal mitoses with an intact spindle assembly checkpoint and that they are able to complete cytokinesis. Our results also exclude DNA rereplication as a major mechanism of polyploidization in E7-expressing cells upon microtubule disruption. Instead, we have shown that while normal cells arrest at the postmitotic checkpoint after adaptation to the spindle assembly checkpoint, E7-expressing cells replicate their DNA and propagate as polyploid cells. Thus, abrogation of the postmitotic checkpoint leads to polyploidy formation in E7-expressing human epithelial cells. Our results suggest that downregulation of pRb is important for E7 to induce polyploidy and abrogation of the postmitotic checkpoint.An important hallmark of human cancers is aneuploidy, the state in which a cell has extra or missing chromosomes (12,25). Polyploidy is the state in which cells have more than two equal sets of chromosomes and is thought to be an early event in multistep carcinogenesis that can lead to aneuploidy (1, 24), as exemplified in Barrett's esophagus (11). Polyploidy has recently been shown to occur as an early event in cervical carcinogenesis and to predispose the cells to aneuploidy (26). Other recent studies have shown that tetraploid but not diploid mouse or human cells induce tumor formation in mice (3, 9). These studies highlight the potential importance of polyploidy in carcinogenesis.The cellular mechanisms responsible for this polyploidy formation are as of yet undetermined, but several models have been proposed. First, abrogation of the spindle assembly checkpoint followed by cleavage failure may lead to polyploidy formation (36,40). A second proposed model is rereplication, a process of multiple rounds of DNA replication without an intervening mitosis. Third, cells that adapt to the mitotic spindle checkpoint halt in a G 1 -like state with 4C DNA content. Abrogation of this postmitotic checkpoint allows the cells to replicate their 4C DNA content, leading to polyploidy formation. This has been shown in cells that express the human papillomavirus type 16 (HPV-16) E6 oncogene that degrades p53 (21). Finally, cleavage failure, which yiel...

The gene encoding for core-binding factor B (CBFB) is altered in acute myeloid leukemia samples with an inversion in chromosome 16, expressing the fusion protein CBFB-SMMHC. Previous studies have shown that this oncoprotein interferes with hematopoietic differentiation and proliferation and participates in leukemia development. In this study, we provide evidence that CbfB modulates the oncogenic function of this fusion protein. We show that CbfB plays an important role in proliferation of hematopoietic progenitors expressing CbfB-SMMHC in vitro. In addition, CbfB-SMMHC-mediated leukemia development is accelerated in the absence of CbfB. These results indicate that the balance between CbfB and CbfB-SMMHC directly affects leukemia development, and suggest that CBF-specific therapeutic molecules should target CBFB-SMMHC function while maintaining CBFB activity.

Probing the active site of rhodanase with disulfide reagents Uses of gel electrophoresis to monitor detergent enzymes: application to safety assessments C-terminal sequence analysis of polypeptides containing C-terminal proline Exploiting automated protein sequence analysis: in situ cleavage and resequencing Study of a rearrangement at the C-terminus of peptides during the collision activated dissociation experiments Analysis of the microheterogeneity of recombinant Schistosoma mansoni antigen rSmp28 reveals N-formyl-alanine as a new post-translational modification in Saccharomyces cerevisiae Characterization of a recombinant cytosolic domain of CD45 phosphatase Preparation and characterization of the site-directed E211Q mutant of yeast enolase 1 Direct mass spectrometric analyses for protein chemistry studies Structure characterization of membrane bound and surface adsorbed protein p58, A membrane-and microfilament-associated gag-like protein from ascites tumor cell microvilli, binds SrC SH3 domain through a poly-proline motif 517 Direct determination of immobilized protein concentration Conformational flexibility of the Gly-Gly dipeptide within protein structures A highly conserved N-terminal sequence for teleost vitellogenins Rapid fingerprinting of proteins with immobilized protease columns Protein, farnesyi transferase. Evidence for an electrophilic substitution mechanism. Disulphide mapping of prolamins Conformational comparison in the snake toxin family Mapping and characterization of proteins associated with morphological transformation in the SHE cell assay Solution structure of a cyclic c~-MSH analogue--initial NMR studies Identification of modified PTH-amino acids in protein sequence analysis Crystallization of a thermal stable trypsin inhibitor from Phaseolus lunatus Characterization of recombinant porin by mass spectrometry Insights into the structure and active-site architecture of IPP:DMAPP isomerase Counting cysteine and cystine residues in proteins and peptides using MALDI-TOF mass spectrometry Detection of di-PTH-cys for assignment of disulfide linkages Mutagenesis of the cyclic AMP receptor protein (CRP) of Escherichia coli MPSA Abstract Listing 519 Reed J. Harris H5. Evaluating protein modification sites observed by N-terminal sequence analysis Detection of low levels of hydroxylysine in non-collagenous proteins Chemical examination of the diphenylphosphoroisothiocyanatidate/trimethylsilnolate method for protein carboxyl-terminal degradations Aptase and molecular chaperone activities of the maize endoplasmic reticulum-resident Stress-70 protein Micropreparation for sequence analysis of peptides of proteins obtained from old archived polyacrylamide gels by in situ trypsin digestion Identification and characterization of metal protein complexes of crude cell extracts by mass-spectrometry The structure and function of plant leginsulin Probing the Mg 2+ binding site of/3-galactosidase (E. coli) In-gel reduction and alkylation of proteins and enzymatic digestion: application to the sequence analysi...

Acute myeloid leukemia (AML) samples with chromosome 16 inversion express the CBFb-MYH11 fusion gene. One of three RUNX genes (RUNX1, RUNX2, and RUNX3) encode the α-subunit and CBFb encodes the β subunit of the heterodimeric transcription factor CBF. This transcription factor is a key regulator of multiple steps on hematopoietic differentiation. Studies in the mouse have determined that Cbfb-MYH11 expression impairs hematopoiesis, and that it induces AML in collaboration with other mutations. To further study the effects of Cbfb-MYH11 expression in hematopoiesis and leukemogenesis, we created a Cbfb-MYH11 conditional knock-in model (Cbfb56M), using the Cre-loxP recombination system. In this model, the floxed Cbfb allele expresses wildtype Cbfb, and the downstream Cbfb-MYH11 is induced upon Cre-mediated deletion. Floxed heterozygous and homozygous Cbfbfb56M mice are disease-free, indicating that Cbfb expressed from the floxed allele is functional. In addition, Cbfb-MYH11 was efficiently induced in over 80% of bone marrow cells from Cbfb56M/+/Mx1-Cre mice after pIpC injection. The preleukemic effects of Cbfb-MYH11 in hematopoiesis were analyzed using induced mice and non-competitive repopulation assays. First, circulating B-cells were reduced soon after Cbfb-MYH11 induction, and a significant differentiation block at the pre-pro B-cell stage was detected in the bone marrow. Second, thymic T-cell differentiation of induced mice showed impairment of DN2 to DN3 stage and reduction of thymic size in 3/10 induced mice analyzed. Interestingly, the number of circulating T cells was unaffected in repopulation assays. Third, platelets were reduced 50% in peripheral blood and megakaryocyte number was reduced in bone marrow. Fourth, we found an expanded abnormal progenitor compartment (Lin-kit+Sca1-) that accumulated in the bone marrow and spleen. In vitro differentiation assays showed a 2-to-3 fold increase of Cbfb-MYH11 colonies when compared to controls. The colony size was smaller, and showing partial differentiation deficiency. Interestingly, the colony numbers declined upon serial plating below controls. Taken together these results indicate that Cbfb-MYH11 induce accumulation of late progenitors (primarily myeloid progenitors) with limited self-renewal potential. Acute myeloid leukemia arised spontaneously 4 to 6 months after Cbfb-MYH11 induction, with expansion of blast- and monoblastic-like leukemic cells defined as Lin-kit+Sca1-. Leukemic mice showed infiltration in several tissues, including spleen, liver, brain, and lungs. To test whether a second “hit” is necessary in this model, we used bone marrow transduction assays to co-express Cbfb-MYH11 and the candidate cooperating gene Runx2. Recipient mice developed AML with similar phenotype 6 to 12 weeks post transplantation, while control mice remained healthy for 6 months. This study demonstrates that Cbfb-MYH11 expression (i) defines a preleukemic stage with hematopoietic differentiation block at stages associated with Runx function, (ii) the accumulation of an abnormal progenitor cell population (Lin-/kit+/Sca1-), (iii) induces additional mutations to efficiently develop AML, and (iv) synergizes with Runx2 in AML development.