1: J Cell Biol. 2005 Nov 7;171(3):471-82. 

MyoD induces myogenic differentiation through cooperation of its NH2- and
COOH-terminal regions.

Ishibashi J, Perry RL, Asakura A, Rudnicki MA.

Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa,
Ontario K1N 6N5, Canada.

MyoD and Myf5 are basic helix-loop-helix transcription factors that play key but
redundant roles in specifying myogenic progenitors during embryogenesis.
However, there are functional differences between the two transcription factors
that impact myoblast proliferation and differentiation. Target gene activation
could be one such difference. We have used microarray and polymerase chain
reaction approaches to measure the induction of muscle gene expression by MyoD
and Myf5 in an in vitro model. In proliferating cells, MyoD and Myf5 function
very similarly to activate the expression of likely growth phase target genes
such as L-myc, m-cadherin, Mcpt8, Runx1, Spp1, Six1, IGFBP5, and Chrnbeta1.
MyoD, however, is strikingly more effective than Myf5 at inducing
differentiation-phase target genes. This distinction between MyoD and Myf5
results from a novel and unanticipated cooperation between the MyoD NH(2)- and
COOH-terminal regions. Together, these results support the notion that Myf5
functions toward myoblast proliferation, whereas MyoD prepares myoblasts for
efficient differentiation.

PMID: 16275751 [PubMed - in process]


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2: Leukemia. 2005 Nov;19(11):1958-68. 

Effects of overexpression of HBP1 upon growth and differentiation of leukemic
myeloid cells.

Yao CJ, Works K, Romagnoli PA, Austin GE.

Department of Pathology and Laboratory Medicine, Veterans Affairs Medical
Center, Decatur, GA 30033, USA.

HMG-box containing protein 1 (HBP1) is a member of the high mobility group (HMG)
of chromosomal proteins. Since HBP1 exhibits tumor-suppressor activity in
nonmyeloid tissues, we examined the effects of ectopic overexpression of HBP1
upon the growth and differentiation of myeloid cells. We prepared transient and
stable transfectants of the myeloblast cell line K562, which overexpress HBP1
mRNA and protein. HBP1 transfectants displayed slower growth in cell culture and
reduced colony formation in soft agar, retardation of S-phase progression,
reduced expression of cyclin D1 and D3 mRNAs and increased expression of p21
mRNA. HBP1 transfectants also underwent increased apoptosis, as demonstrated by
morphology and binding of Annexin V. Fas ligand mRNA levels were increased in
HBP1 transfectants, suggesting involvement of the Fas/Fas ligand pathway. HBP1
overexpression enhanced differentiation of K562 cells towards erythroid and
megakaryocyte lineages, as evidenced by increased hemoglobin and CD41a
expression. Overexpression of HBP1 modulated mRNA levels for myeloid-specific
transcription factors C/EBPalpha, c-Myb, c-Myc, and JunB, as well as
lineage-specific transcription factors PU.1, GATA-1, and RUNX1. These findings
suggest that in myeloid cells HBP1 may serve as a tumor suppressor and a general
differentiation inducer and may synergize with chemical differentiating agents
to enhance lineage-specific differentiation.

PMID: 16179914 [PubMed - indexed for MEDLINE]


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3: Leukemia. 2005 Jun;19(6):1042-50. 

Gene expression profiling of leukemic cell lines reveals conserved molecular
signatures among subtypes with specific genetic aberrations.

Andersson A, Eden P, Lindgren D, Nilsson J, Lassen C, Heldrup J, Fontes M, Borg
A, Mitelman F, Johansson B, Hoglund M, Fioretos T.

Department of Clinical Genetics, Lund University Hospital, SE-221 85 Lund,
Sweden. anna.andersson@klingen.lu.se

Hematologic malignancies are characterized by fusion genes of
biological/clinical importance. Immortalized cell lines with such aberrations
are today widely used to model different aspects of leukemogenesis. Using cDNA
microarrays, we determined the gene expression profiles of 40 cell lines as well
as of primary leukemias harboring 11q23/MLL rearrangements, t(1;19)[TCF3/PBX1],
t(12;21)[ETV6/RUNX1], t(8;21)[RUNX1/CBFA2T1], t(8;14)[IGH@/MYC],
t(8;14)[TRA@/MYC], t(9;22)[BCR/ABL1], t(10;11)[PICALM/MLLT10],
t(15;17)[PML/RARA], or inv(16)[CBFB/MYH11]. Unsupervised classification revealed
that hematopoietic cell lines of diverse origin, but with the same primary
genetic changes, segregated together, suggesting that pathogenetically important
regulatory networks remain conserved despite numerous passages. Moreover,
primary leukemias cosegregated with cell lines carrying identical genetic
rearrangements, further supporting that critical regulatory pathways remain
intact in hematopoietic cell lines. Transcriptional signatures correlating with
clinical subtypes/primary genetic changes were identified and annotated based on
their biological/molecular properties and chromosomal localization. Furthermore,
the expression profile of tyrosine kinase-encoding genes was investigated,
identifying several differentially expressed members, segregating with primary
genetic changes, which may be targeted with tyrosine kinase inhibitors. The
identified conserved signatures are likely to reflect regulatory networks of
importance for the transforming abilities of the primary genetic changes and
offer important pathogenetic insights as well as a number of targets for future
rational drug design.

PMID: 15843827 [PubMed - indexed for MEDLINE]


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4: J Biol Chem. 2004 Apr 9;279(15):15678-87. Epub 2004 Jan 27. 

AML1/RUNX1 increases during G1 to S cell cycle progression independent of
cytokine-dependent phosphorylation and induces cyclin D3 gene expression.

Bernardin-Fried F, Kummalue T, Leijen S, Collector MI, Ravid K, Friedman AD.

Division of Pediatric Oncology, Johns Hopkins University, Baltimore, Maryland
21231, USA.

AML1/RUNX1, a member of the core binding factor (CBF) family stimulates
myelopoiesis and lymphopoiesis by activating lineage-specific genes. In
addition, AML1 induces S phase entry in 32Dcl3 myeloid or Ba/F3 lymphoid cells
via transactivation. We now found that AML1 levels are regulated during the cell
cycle. 32Dcl3 and Ba/F3 cell cycle fractions were prepared using elutriation.
Western blotting and a gel shift/supershift assay demonstrated that endogenous
CBF DNA binding and AML1 levels were increased 2-4-fold in S and G(2)/M phase
cells compared with G(1) cells. In addition, G(1) arrest induced by mimosine
reduced AML1 protein levels. In contrast, AML1 RNA did not vary during cell
cycle progression relative to actin RNA. Analysis of exogenous Myc-AML1 or
AML1-ER demonstrated a significant reduction in G(1) phase cells, whereas levels
of exogenous DNA binding domain alone were constant, lending support to the
conclusion that regulation of AML1 protein stability contributes to cell cycle
variation in endogenous AML1. However, cytokine-dependent AML1 phosphorylation
was independent of cell cycle phase, and an AML1 mutant lacking two ERK
phosphorylation sites was still cell cycle-regulated. Inhibition of AML1
activity with the CBFbeta-SMMHC or AML1-ETO oncoproteins reduced cyclin D3 RNA
expression, and AML1 bound and activated the cyclin D3 promoter. Signals
stimulating G(1) to S cell cycle progression or entry into the cell cycle in
immature hematopoietic cells might do so in part by inducing AML1 expression,
and mutations altering pathways regulating variation in AML1 stability
potentially contribute to leukemic transformation.

PMID: 14747476 [PubMed - indexed for MEDLINE]


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5: Cancer Res. 2003 Oct 1;63(19):6144-8. 

Inhibition of the translocated c-myc in Burkitt's lymphoma by a PNA
complementary to the E mu enhancer.

Cutrona G, Carpaneto EM, Ponzanelli A, Ulivi M, Millo E, Scarfi S, Roncella S,
Benatti U, Boffa LC, Ferrarini M.

Division of Medical Oncology C, National Cancer Research Institute, Istituto
Superiore di Sanita, Largo R. Benzi 10, 16132 Genoa, Italy.
giovanna.cutrona@istge.it

In Burkitt's Lymphoma there is an up-regulation of the c-myc oncogene caused by
its translocation from chromosome 8 to chromosome 14, often close to the E mu
enhancer of the immunoglobulin heavy chain locus (IgH). In Burkitt's Lymphoma
cells, a peptide nucleic acid complementary for a specific unique E mu intronic
sequence selectively blocked the expression of the c-myc oncogene under E mu
control but not of other c-myc alleles. This Peptide Nucleic Acid also inhibited
IgM expression in B cells. The finding that PNAs specific for a regulatory
noncoding sequence can block gene expression has important conceptual and
practical implications.

PMID: 14559793 [PubMed - indexed for MEDLINE]


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6: Blood Cells Mol Dis. 2003 Mar-Apr;30(2):194-200. 

The Runx genes as dominant oncogenes.

Cameron ER, Blyth K, Hanlon L, Kilbey A, Mackay N, Stewart M, Terry A, Vaillant
F, Wotton S, Neil JC.

Molecular Oncology Laboratory, Institute of Comparative Medicine, University of
Glasgow Veterinary School, Glasgow G61 1QH, UK. E.R.Cameron@vet.gla.ac.uk

We have shown previously that Runx2 is a frequent target (approximately equal to
30%) for proviral insertion in murine leukemia virus (MLV) induced T cell tumors
in CD2-MYC transgenic mice. Further investigation of a large panel of these
tumors revealed that a small number also contain insertions at either Runx3 or
Runx1. None of the tumors contained insertions at more than one family member,
but in each case proviral insertion was associated with a high level of
expression from the upstream (P1) promoter of the respective target gene.
Moreover, we confirmed that transcriptional activation of Runx1 does not affect
the integrity of the coding sequence, as previously observed for Runx2. These
observations suggest that the three Runx genes act as functionally redundant
oncogenes in T-cell lymphoma development. To explore the oncogenic potential of
Runx2 further we created transgenic mice that over-express this gene in the T
cell compartment. These CD2-Runx2 animals show a preneoplastic enlargement of
the CD8 immature single positive (ISP) thymocyte pool and develop lymphomas at a
low incidence. Although the CD8 ISP population is greatly increased, unlike
their wild type counterparts these cells are largely non-cycling. Co-expression
of c-MYC in this lineage accentuates the CD8 ISP skew and induces rapid tumor
development, confirming the potent synergy that exists between these two
oncogenes. Experiments designed to understand the nature of the observed synergy
are ongoing and are based on the hypothesis that Runx2 may exert a survival
effect in c-MYC expressing tumors in vivo while c-MYC may rescue cells from the
antiproliferative effects of Runx2. The oncogenic potential of Runx1 is also
being assessed using primary murine embryonic fibroblasts (MEFs). These studies
have revealed that while Runx1 exerts a growth suppressive effect in wild type
cells a growth promoting effect is seen in the absence of p53, suggesting that
the Runx genes may harbor latent oncogene-like properties.

Publication Types:
    Review
    Review, Tutorial

PMID: 12732183 [PubMed - indexed for MEDLINE]


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7: Cancer Res. 2002 Dec 15;62(24):7181-5. 

Proviral insertion indicates a dominant oncogenic role for Runx1/AML-1 in T-cell
lymphoma.

Wotton S, Stewart M, Blyth K, Vaillant F, Kilbey A, Neil JC, Cameron ER.

Molecular Oncology Laboratory, Institute of Comparative Medicine, University of
Glasgow Veterinary School, United Kingdom.

The RUNX1/AML1 gene is a frequent target for chromosomal translocations in human
leukemia. The biological properties of the resulting fusion products and the
finding that haploinsufficiency increases the risk of developing leukemia (W-J.
Song et al., Nat. Genet., 23: 166-175, 1999; M. Osata et al., Blood, 93:
1817-1824, 1999) have led to the widely held view that RUNX1 loss-of-function is
a key event. However, we now report that the gene is a target for insertional
mutagenesis in T-cell lymphomas of mice carrying a MYC oncogene, where promoter
insertion results in overexpression without affecting the integrity of the
coding sequence. Moreover, Runx1 haploinsufficiency does not accelerate lymphoma
development in MYC/Runx2 transgenic or murine leukemia virus-infected mice.
These findings reveal that the Runx1 gene can also act as a dominant oncogene
and suggest that the involvement of the Runx gene family in human leukemia may
be more widespread and complex than previously realized.

PMID: 12499254 [PubMed - indexed for MEDLINE]


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8: Cancer Biol Ther. 2002 Sep-Oct;1(5):492-6. 

c-Myc overcomes cell cycle inhibition by CBFbeta-SMMHC, a myeloid leukemia
oncoprotein.

Bernardin F, Yang Y, Civin CI, Friedman AD.

Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 1650 Orleans Street,
Baltimore, MD 21231, USA.

Thirty percent of acute myeloid leukemia cases express a Core Binding Factor
(CBF) oncoprotein or harbor point mutations in one or both AML1 (RUNX1) genes.
Each of these alterations reduces endogenous CBF activities. CBFbeta-SMMHC is
expressed from the inv(16) chromosome in 8% of AML cases and inhibits endogenous
CBF DNA-binding. Inhibition of CBF reduces Retinoblastoma protein
phosphorylation and slows the G(1) to S cell cycle transition. c-Myc, a protein
which stimulates S phase entry, is over-expressed in one-third of AMLs. We have
developed Ba/F3 cell lines in which zinc regulates CBFbeta-SMMHC expression and
4-hydroxytamoxifen activates c-Myc-ER. In these lines, c-Myc-ER overcomes
inhibition of cell cycle progression mediated by CBFbeta-SMMHC. CBFbeta-SMMHC
does not affect endogenous c-Myc RNA levels, indicating that CBF does not
regulate the c-Myc gene. Conversely, c-Myc-ER does not alter CBF DNA-binding
activity. Thus, c-Myc-ER acts downstream of CBFbeta-SMMHC to stimulate cell
cycle progression. In a subset of CBF leukemias, elevated expression of c-Myc is
expected to facilitate the proliferation of the leukemic blasts and thereby
potentiate the ability of CBF oncoproteins to block differentiation.

PMID: 12496475 [PubMed - indexed for MEDLINE]


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9: J Virol. 1997 Jul;71(7):5080-7. 

Stability of AML1 (core) site enhancer mutations in T lymphomas induced by
attenuated SL3-3 murine leukemia virus mutants.

Amtoft HW, Sorensen AB, Bareil C, Schmidt J, Luz A, Pedersen FS.

Department of Molecular and Structural Biology, University of Aarhus, Aarhus C,
Denmark.

Murine retrovirus SL3-3 is highly T lymphomagenic. Its pathogenic properties are
determined by the transcriptional enhancer of the U3 repeat region which shows
preferential activity in T cells. Within the U3 repeats, the major determinant
of T-cell specificity has been mapped to binding sites for the AML1
transcription factor family (also known as the core binding factor [CBF],
polyomavirus enhancer binding protein 2 [PEBP2], and SL3-3 enhancer factor 1
[SEF-1]). SL3-3 viruses with AML1 site mutations have lost a major determinant
of T-cell-specific enhancer function but have been found to retain a
lymphomagenic potential, although disease induction is slower than for the SL3-3
wild type. To compare the specificities and mechanisms of disease induction of
wild-type and mutant viruses, we have examined lymphomas induced by mutant
viruses harboring transversions of three consecutive base pairs critical to AML1
site function (B. Hallberg, J. Schmidt, A. Luz, F. S. Pedersen, and T.
Grundstrom. J. Virol. 65:4177-4181, 1991). Our results show that the mutated
AML1 sites are genetically stable during lymphomagenesis and that ecotropic
provirus numbers in DNA of tumors induced by wild-type and mutant viruses fall
within the same range. Moreover, proviruses were found to be integrated at the
c-myc locus in similar proportions of wild-type and mutant SL3-3-induced tumors,
and the mutated AML1 sites of proviruses at c-myc are unaltered. In some cases,
however, including one c-myc-integrated provirus, a single-base pair change was
detected in a second, weaker AML1 binding site. By DNA rearrangement analysis of
the T-cell receptor beta-locus, tumors induced by the AML1 site mutants are
found to be of the T-cell type. Thus, although the AML1 site mutants have
weakened T-cell-specific enhancers they are T-lymphomagenic, and wild-type- and
mutant-virus-induced tumor DNAs are similar with respect to the number of
overall ecotropic and c-myc-integrated clonal proviruses. The SL3-3 wild-type
and AML1 site mutant viruses may therefore induce disease by similar mechanisms.

PMID: 9188573 [PubMed - indexed for MEDLINE]


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