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Acute promyelocytic leukemia (APL) is characterized by specific chromosomal translocations, which generate fusion proteins such as promyelocytic leukemia (PML)-retinoic acid receptor (RAR)alpha and promyelocytic leukemia zinc finger (PLZF)-RARalpha (X-RARalpha). In this study, we have applied lac operator array systems to study the effects of X-RARalpha versus wild-type RARalpha on large-scale chromatin structure. The targeting of these enhanced cyan fluorescent protein-lac repressor-tagged RARalpha-containing proteins to the gene-amplification chromosomal region by lac operator repeats led to local chromatin condensation, recruitment of nuclear receptor corepressor, and histone deacetylase complex. The addition of retinoic acid (RA) induced large-scale chromatin decondensation in cells expressing RARalpha; however, cells expressing X-RARalpha, especially PML-RARalpha, demonstrated insensitive response to this effect of all-trans retinoic acid (ATRA). Although we did not reveal differences in RA-dependent colocalization of either silencing mediator for retinoid and thyroid or steroid receptor coactivator (SRC)-1 with RARalpha versus X-RARalpha, the hormone-independent association between SRC-1 and X-RARalpha on the array has been identified. Rather, compared with cells expressing RARalpha, fluorescence recovery after photobleaching of live transfected cells, demonstrated decreased mobility of SRC-1 on the X-RARalpha-bound chromatin. Thus, the impaired ability of APL fusion proteins to activate gene transcription in response to ATRA corresponds to their reduced ability to remodel chromatin, which may link to their ability to impair the mobility of key nuclear receptor coregulators.

While formation of higher-order oncogenic transcriptional complexes is critical for RARalpha fusion proteins in acute promyelocytic leukemia, the essential components and their roles in mediating transformation are still largely unknown. To this end, the present study demonstrates that homodimerization is not sufficient for RARalpha fusion-mediated transformation, which requires higher-order homotetramerization. Surprisingly, intrinsic homo-oligomeric DNA binding by the fusion proteins is also dispensable. Importantly, higher-order RXR/RARalpha fusion hetero-oligomeric complexes that aberrantly recruit transcriptional corepressors to downstream targets are essential for transformation. Intervention of RXR-dependent pathways by panRXR-agonists or RXRalpha shRNAs suppresses RARalpha fusion-mediated transformation. Taken together, these results define the oncogenic threshold for self-association and reveal the pathological significance of higher-order RARalpha fusion/RXR hetero-oligomeric complexes and their potential value as a therapeutic target.

Almost 100% of APL patients carry chimeric transcripts encoding truncated RARalpha fused to homo-oligomerization domains from partner proteins. To gain further insights into the cellular transformation mechanisms mediated by RARalpha fusion proteins, thorough structure/function analyses have been performed and identified the POZ homo-oligomerization domain as the minimal transformation domain that is necessary and sufficient for PLZF-RARalpha-mediated in vitro transformation of primary hematopoietic cells. A transformation-incompetent PLZF-RARalpha mutant defective in homo-oligomerization but not corepressor interaction could be rescued by synthetic FKBP-oligomerization domains. Furthermore, an artificial FKBP-RARalpha construct not only mimicked various biochemical properties of bona fide RARalpha fusion proteins but also mediated an ATRA-dependent transformation. Taken together, these findings endorse an oligomerization-dependent mechanism for RARalpha-mediated transformation and suggest a potential avenue for molecular therapy.

Active Shape Model (ASM) is a popular statistical shape model for objects localization. It combines a Point Distribution Model (PDM) and local grey-level model of each landmark point to achieve reliable and accurate result in image interpretation. However, in 2D images, the one-dimensional profile grey-level models will lose information in other directions. In this paper, we modeled the grey-level information of the k x k square neighbor region around each point, hoping to take more information into account. Yet, further works should be done in order to compare the searching results obtained by square grey-level models with those by one-dimensional profile grey-level models.

The thin sectioning has been widely applied in electron microscopy (EM), and successfully used for an in situ observation of inner ultrastructure of cells. This powerful technique has recently been extended to the research field of atomic force microscopy (AFM). However, there have been no reports describing AFM imaging of serial thin sections and three-dimensional (3-D) reconstruction of cells and their inner structures. In the present study, we used AFM to scan serial thin sections approximately 60 nm thick of a mouse embryonic stem (ES) cell, and to observe the in situ inner ultrastructure including cell membrane, cytoplasm, mitochondria, nucleus membrane, and linear chromatin. The high-magnification AFM imaging of single mitochondria clearly demonstrated the outer membrane, inner boundary membrane and cristal membrane of mitochondria in the cellular compartment. Importantly, AFM imaging on six serial thin sections of a single mouse ES cell showed that mitochondria underwent sequential changes in the number, morphology and distribution. These nanoscale images allowed us to perform 3-D surface reconstruction of interested interior structures in cells. Based on the serial in situ images, 3-D models of morphological characteristics, numbers and distributions of interior structures of the single ES cells were validated and reconstructed. Our results suggest that the combined AFM and serial-thin-section technique is useful for the nanoscale imaging and 3-D reconstruction of single cells and their inner structures. This technique may facilitate studies of proliferating and differentiating stages of stem cells or somatic cells at a nanoscale.

Acute promyelocytic leukemia (APL) cells contain one of five chimeric retinoic acid alpha-receptor (RAR alpha) genes (X-RAR alpha) created by chromosomal translocations or deletion; each generates a fusion protein thought to transcriptionally repress RAR alpha target genes and block myeloid differentiation by an incompletely understood mechanism. To gain spatiotemporal insight into these oncogenic processes, we employed fluorescence microscopy and fluorescence recovery after photobleaching (FRAP). Fluorescence microscopy demonstrated that the intracellular localization of each of the X-RAR alpha proteins was distinct from that of RAR alpha and established which portion(s) of each X-RAR alpha protein-X, RAR, or both-contributed to its altered localization. Using FRAP, we demonstrated that the intranuclear mobility of each X-RAR alpha was reduced compared to that of RAR alpha. In addition, the mobility of each X-RAR alpha was reduced further by ligand addition, in contrast to RAR alpha, which showed no change in mobility when ligand was added. Both the reduced baseline mobility of X-RAR alpha and the ligand-induced slowing of X-RAR alpha could be attributed to the protein interaction domain contained within X. RXR alpha aberrantly colocalized within each X-RAR alpha; colocalization of RXR alpha with promyelocytic leukemia (PML)-RAR alpha resulted in reduced mobility of RXR alpha. Thus, X-RAR alpha may interfere with RAR alpha through its aberrant nuclear dynamics, resulting in spatial and temporal sequestration of RXR alpha and perhaps other nuclear receptor coregulators critical for myeloid differentiation.

Acute promyelocytic leukemia (APL) is a form of acute myelogenous leukemia characterized by chromosomal alterations involving the retinoic acid receptor-alpha (RARalpha) gene that generate unique chimeric proteins (N-RARalpha) and by clinical responsiveness to all-trans-retinoic acid (ATRA) treatment. APL cells are notable for differentiation block and resistance to apoptosis. While increasing evidence suggests that N-RARalpha fusion proteins interfere with normal RARalpha transcription function at retinoic acid response elements (RAREs) resulting in inhibition of normal myeloid differentiation, the mechanism for apoptosis resistance remains unexplained. Recently, we and others have reported that APL-fusion proteins can augment STAT3 transcriptional activity. Constitutive STAT3 activation has been observed in a number of hematopoietic and non-hematopoietic malignancies where it contributes to apoptosis resistance. In this review, we summarize a series of recent observations concerning cross talk between the retinoic acid and STAT3 signaling pathways in APL cells. These findings support the hypothesis that apoptosis resistance in APL may be mediated through the effects of APL fusion proteins on STAT3 signaling and suggest that targeting of STAT3 may be a useful adjunctive treatment strategy in APL.

Nuclear mitotic apparatus protein-retinoic acid receptor alpha (NuMA-RARalpha) is the fourth of five fusion proteins identified in acute promyelocytic leukemia (APL) patients. The molecular basis for its oncogenic activity has not been delineated. In gel-shift assays, NuMA-RARalpha bound to retinoic acid response elements (RAREs) both as a homodimer and as a heterodimer with RXRalpha. The binding profile of NuMA-RARalpha to a panel of RAREs was very similar to PML-RARalpha and PLZF-RARalpha. In transient transfection assays using HepG2 cells, NuMA-RARalpha inhibited wild-type RARalpha transcriptional activity, while it augmented STAT3 transcriptional activity. In GST-pull down experiments, NuMA-RARalpha formed a complex with the corepressor SMRT, was released from the NuMA-RARalpha/SMRT complexes by all-trans retinoic acid (ATRA) at 10(-7)-10(-6) M and became associated with the coactivator TRAM-1 at 10(-8) M ATRA. Studies comparing NuMA-RARalpha with NuMA-RARalpha(deltaCC) demonstrated that the dimerization or alpha-helical coiled-coil domain of NuMA was required for homodimer formation, transcriptional repression of wild-type RARalpha, transcriptional activation of STAT3, and stability of the NuMA-RARalpha/SMRT complex. Confocal fluorescent microscopy of HeLa cells was performed following transient expression of cyan fluorescent protein (CFP)-tagged proteins and incubation of cells with or without ATRA. Within the nucleus, CFP-NuMA-RARalpha exhibited a speckled pattern identical to that observed in cells transfected with CFP-NuMA. Furthermore, CFP-NuMA-RARalpha colocalized with yellow fluorescent protein-tagged (YFP)-NuMA. In contrast, CFP-NuMA-RARalpha(deltaCC) exhibited a diffuse granular pattern within the nucleus, similar to RARalpha. These results indicate that the dimerization domain of NuMA-RARalpha is critical for each of the known oncogenic activities of NuMA fusion proteins as well as its sequestration to nuclear sites normally occupied by NuMA and is distinct from RARalpha.

We describe here two mouse mutants, yellow submarine (Ysb) and light coat and circling (Lcc). Ysb arose as the result of insertions of a transgene, pAA2, into the genome. Lcc is an independent, radiation-induced mutation. Both mutants are characterized by recessive circling behavior and deafness, associated with a non-segregating, semi-dominant yellow coat color. Complementation tests showed that Ysb and Lcc are allelic. We attribute the yellow coat in Ysb and Lcc mice to the absence of black awl overhairs, increased agouti zigzag underhairs, and the presence of agouti awls with long subapical yellow pigment. Chromosomal mapping and genomic characterization showed the Ysb and Lcc mutations involve complex chromosomal rearrangements in overlapping regions of mouse chromosome 3, A2/A3-B/C and B-E1, respectively. Ysb and Lcc show for the first time, to our knowledge, the presence of genes in the B-C region of chromosome 3 important for balance and hearing and the pigmentation and specification of coat hair., ((c)2002 Elsevier Science (USA).)

The physiologic actions of retinoic acids (RAs) are mediated through RA receptors (RARs) and retinoid X receptors (RXRs). The RAR(alpha) gene has drawn particular attention because it is the common target in all chromosomal translocations in acute promyelocytic leukemia (APL), a unique model in cancer research that responds to the effect of RA. In the great majority of patients with APL, RAR(alpha) is fused to the PML gene as a result of the t(15;17) translocation. Three distinct types of PML-RAR(alpha) transcripts, long (L), short (S), and variant (V), were identified. The V-type is characterized by truncation of exon 6 of PML and in some cases by the insertion of a variable "spacer" sequence between the truncated PML and RAR(alpha) mRNA fusion partners, although the precise mechanisms underlying formation of the V-type transcript remain unclear. To get further insights into the molecular basis of the t(15;17), we sequenced the entire genomic DNA region of RAR(alpha). Of note, all previously reported "spacer" sequences in V-type transcripts were found in intron 2 of the RAR(alpha) gene and most of these sequences were flanked by gt splice donor sites. In most cases, these "cryptic" coding sequences maintained the ORF of the chimeric transcript. Interestingly, two cases with a relatively long spacer sequence showed APL cellular and clinical resistance to RA treatment. In these cases, the aberrant V-type PML-RAR(alpha) protein displayed increased affinity to the nuclear corepressor protein SMRT, providing further evidence that RA exerts the therapeutic effect on APL through modulation of the RAR-corepressor interaction. Finally, among patients with the L- or S-type PML-RAR(alpha) fusion transcript, some consensus motifs were identified at the hotspots of the chromosome 17q breakpoints within intron 2 of RAR(alpha), strengthening the importance of this intron in the molecular pathogenesis of APL.

Signal transducer and activator of transcription (STAT) 5b-retinoic acid receptor (RAR) alpha is the fifth fusion protein identified in acute promyelocytic leukemia (APL). Initially described in a patient with all-trans retinoic acid (ATRA)-unresponsive disease, STAT5b-RARalpha resulted from an interstitial deletion on chromosome 17. To determine the molecular mechanisms of myeloid leukemogenesis and maturation arrest in STAT5b-RARalpha(+) APL and its unresponsiveness to ATRA, we examined the effect of STAT5b-RARalpha on the activity of myeloid transcription factors including RARalpha/retinoid X receptor (RXR) alpha, STAT3, and STAT5 as well as its molecular interactions with the nuclear receptor corepressor, SMRT, and nuclear receptor coactivator, TRAM-1. STAT5b-RARalpha bound to retinoic acid response elements (RAREs) both as a homodimer and as a heterodimer with RXRalpha and inhibited wild-type RARalpha/RXRalpha transactivation. Although STAT5b-RARalpha had no effect on ligand-induced STAT5b activation, it enhanced interleukin 6-induced STAT3-dependent reporter activity, an effect shared by other APL fusion proteins including promyelocytic leukemia-RARalpha and promyelocytic leukemia zinc finger (PLZF)-RARalpha. SMRT was released from STAT5b-RARalpha/SMRT complexes by ATRA at 10(-6) M, whereas TRAM-1 became associated with STAT5b-RARalpha at 10(-7) M. The coiled-coil domain of STAT5b was required for formation of STAT5b-RARalpha homodimers, for the inhibition of RARalpha/RXRalpha transcriptional activity, and for stability of the STAT5b-RARalpha/SMRT complex. Thus, STAT5b-RARalpha contributes to myeloid maturation arrest by binding to RARE as either a homodimer or as a heterodimer with RXRalpha resulting in the recruitment of SMRT and inhibition of RARalpha/RXRalpha transcriptional activity. In addition, STAT5b-RARalpha and other APL fusion proteins may contribute to leukemogenesis by interaction with the STAT3 oncogene pathway.