Phosphorylation regulates in vivo interaction and molecular targeting of serine/arginine-rich pre-mRNA splicing factors.

The SR superfamily of splicing factors and regulators is characterized by arginine/serine (RS)-rich domains, which are extensively modified by phosphorylation in cells. In vitro binding studies revealed that RS domain-mediated protein interactions can be differentially affected by phosphorylation. Taking advantage of the single nonessential SR protein-specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions in vivo using the two-hybrid assay. Strikingly, all RS domain-mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein-specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro. To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Delta yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation. Using a transcriptional repression assay, we further showed that most LexA-SR fusion proteins depend on Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting that molecular targeting of RS domain-containing proteins within the nucleus was also affected. Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.

Proto-oncogene PML controls genes devoted to MHC class I antigen presentation.

Fragments of foreign antigens associated with class I molecules of the major histocompatibility complex (MHC) are presented at the cell surface to elicit an immune response. This presentation requires the coordinated expression of several genes contained in the MHC, including those encoding the MHC class I heavy chain, the proteins LMP-2 and LMP-7, which are involved in the proteasomal degradation of cytosolic antigens into peptide fragments that are destined for association with MHC class I molecules, and TAP-1 and TAP-2, which transport these fragments across the membrane of the endoplasmic reticulum at the start of their journey to the cell surface. In many virus-transformed cell lines and spontaneous tumours, these genes are simultaneously repressed. However, the key factor(s) that are essential for their expression and repression have not been identified. Here we report that the proto-oncogene product PML induces expression of LMP-2, LMP-7, TAP-1 and TAP-2 in an MHC-class I-negative, recurrent tumour, leading to the re-expression of cell-surface MHC in tumours and to rejection of the tumours. PML also regulates MHC expression in untransformed fibroblasts. We conclude that malfunction of PML may enable a tumour to evade the immune defence of its host.

Localization of nascent RNA and CREB binding protein with the PML-containing nuclear body.

The cellular role of the PML-containing nuclear bodies also known as ND10 or PODs remains elusive despite links to oncogenesis and viral replication. Although a potential role in transcription has been considered, direct evidence has been lacking. By developing a novel in vivo nucleic acid labeling approach, we demonstrate the existence of nascent RNA polymerase II transcripts within this nuclear body. In addition, PML and the transactivation cofactor, CREB binding protein (CBP), colocalize within the nucleus. Furthermore, we show that CBP in contrast to PML is distributed throughout the internal core of the structure. Collectively, these findings support a role for this nuclear body in transcriptional regulation.

SRPK2: a differentially expressed SR protein-specific kinase involved in mediating the interaction and localization of pre-mRNA splicing factors in mammalian cells.

Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain-containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain-containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH2 terminus, and a recent study showed that this NH2-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.

Rapid diagnosis of acute promyelocytic leukemia by immunohistochemical localization of PML/RAR-alpha protein.

Acute promyelocytic leukemia (APL) is characterized by a consistent chromosomal aberration that fuses the retinoic acid receptor alpha (RAR alpha) gene with the novel gene PML, resulting in the expression of a PML/RAR-alpha fusion protein. Immunohistochemical examination of APL cells shows a unique abnormal distribution of anti-PML and anti-RAR alpha antibody labeling. The PML labeling pattern observed in normal cells consists of 5 to 10 discrete spherical nuclear bodies called PODs (for "PML oncogenic domains"), whereas that of APL consists of a smaller and far more numerous speckled pattern. We examined malignant cells from patients with a variety of hematopoietic cancers by immunohistochemistry (IH) and found this abnormal PML pattern expressed in cells from patients with t(15;17)-associated leukemia but not in patients with other neoplastic disorders. IH results agreed with reverse transcription polymerase chain reaction for PML/RAR-alpha in 31 of 32 patients with acute myelogenous leukemia, including 5 of 5 patients in whom the initial clinical diagnosis of APL was not supported by cytogenetics, molecular tests, or response to all-trans retinoic acid (RA). Cells from patients with APL were examined during the course of retinoid therapy and at the time of complete remission and relapse. Reorganization of the PML labeling into PODs with normal appearance was observed in cells from patients who received RA. IH showed primarily normal PML staining during clinical remission, although the APL-specific labeling pattern was again seen in cells taken from patients at the time of relapse. Thus, IH provides an independent assay for the presence and expression of the molecular rearrangement of APL. The relative ease and speed of detecting the APL-specific PML labeling pattern should make IH a useful diagnostic tool to guide specific therapy of APL, and establish a direct assay for PML/RAR-alpha protein expression and localization in individual patient cells.

A novel macromolecular structure is a target of the promyelocyte-retinoic acid receptor oncoprotein.

Acute promyelocytic leukemia (APL) is associated with a t(15;17) translocation that creates the promyelocyte-retinoic acid receptor alpha (PML-RAR alpha) fusion gene. Immunohistochemistry demonstrates that PML is a part of a novel macromolecular organelle (including at least three other nuclear proteins) referred to as PML oncogenic domains (PODs). In APL cells, the POD is disrupted into a microparticulate pattern as a consequence of the expression of the PML-RAR oncoprotein. RA treatment of APL cells triggers a reorganization of PML to generate normal-appearing PODs. We propose that PML-RAR is a dominant negative oncoprotein that exerts its putative leukomogenic effect by inhibiting assembly of the POD. According to this proposal, not only is the POD a novel structure, but it can be ascribed an imputed function such that its disruption leads to altered myeloid maturation; this may represent a novel oncogenic target.

Multiple retinoid-responsive receptors in a single cell: families of retinoid "X" receptors and retinoic acid receptors in the Xenopus egg.

In a search for nuclear hormone receptors expressed in early development we found that Xenopus laevis eggs contain mRNAs from two retinoic acid receptor genes (xRAR alpha and xRAR gamma) and two retinoid "X" receptor genes (xRXR alpha and xRXR gamma). We also show that RXRs are members of a family of at least three genes, thus expanding the number of genes encoding retinoic acid-responsive transcription factors to six. With the exception of xRXR gamma, these maternal mRNAs are degraded before gastrulation. The RXRs isolated are differentially activated by retinoic acid and by 3,4-didehydroretinoic acid. Considered together, these four receptors provide a molecular basis for the pleiotropic effects of retinoic acid on early development, and their pattern of expression suggests a role for retinoic acid at the earliest stages of embryonic determination.

Retinoid X receptor-COUP-TF interactions modulate retinoic acid signaling.

We have recently described the properties of direct repeats (DRs) of the half-site AGGTCA as hormone response elements (HREs). According to our results, spacing the half sites by 3, 4, or 5 nucleotides determines specificity of response for vitamin D3, thyroid hormone, and retinoic acid receptors, respectively. This so-called 3-4-5 rule led to the prediction that remaining spacing options of 0, 1, and 2 might serve as targets for other nuclear receptors. A concurrent prediction is that receptors recognizing common sites might display more complex or combinatorial interactions. In exploring these predictions, we discovered that both the retinoid X receptor (RXR) and COUP-TF bind preferentially to a DR-1 motif. In vivo, RXR and COUP-TF display antagonistic action such that RXR-mediated activation is fully repressed by COUP-TF. In vitro studies reveal that COUP-TF and RXR form heterodimers on DR-1. Thus, these results support a general proposal in which the half-site spacing preferences may be used as a means to decipher potentially complex and interactive regulatory circuits.

9-cis retinoic acid is a high affinity ligand for the retinoid X receptor.

All-trans retinoic acid (RA) has previously been shown to modulate the transcriptional properties of the retinoic acid receptor (RAR) and retinoid X receptor (RXR). The inability of all-trans RA to bind to RXR suggests that it may be metabolized to a more active high affinity ligand. We report here an experimental approach that has identified 9-cis RA as an RXR ligand. It is up to 40-fold more potent than all-trans RA in transfection assays and binds with high affinity. The production of 9-cis RA in cultured cells and the identification of this molecule in liver and kidney demonstrates the existence of this molecule in living organisms. The discovery of this novel hormone points to the key role retinoid metabolism may have in generating new signaling pathways.

Nuclear receptor that identifies a novel retinoic acid response pathway.

Molecular cloning and transcriptional activation studies have revealed a new protein similar to the steroid hormone receptors and which responds specifically to vitamin A metabolites. This protein is substantially different in primary structure and ligand specificity from the products of the previously described retinoic acid receptor gene family. By indicating the existence of an additional pathway through which retinoic acid may exert its effects, these data lead to a re-evaluation of retinoid physiology.

Molecular rearrangements of chromosome 22 in chronic myeloid leukemia in a multi-ethnic Malaysian population.

Rearrangements in the DNA of chronic myelogenous leukemia patients of Chinese, Malay and Indian origin were detected in the breakpoint cluster region of chromosome 22 using molecular techniques. The DNA of fifty patients was examined using a 1.2 kb DNA probe. Rearrangements were detected in 46/50 patients. Karyotypic data were available in nine patients, all of whom were Philadelphia chromosome positive and exhibited DNA rearrangement. Detection of the Philadelphia translocation by molecular methods, at this institution, where cytogenetics is not routinely performed, confirms its diagnostic value. The rearrangement data obtained in this study is consistent with molecular features of chronic myelogenous leukemia patients of Western countries.

Clinical stage of chronic granulocytic leukaemia and BCR breakpoint location in South-East Asian patients.

Forty-six Malaysian patients with chronic granulocytic leukaemia were found to be rearranged in the breakpoint cluster region (BCR) of chromosome 22, molecular evidence of Philadelphia chromosome (t9.22) translocation. Through the use of a 1.2 kb 3' BCR probe and two restriction enzyme digests, patients' breakpoints could be localized either to 5' or 3' regions of the BCR. Breakpoint site localization at the time of DNA sampling did not show any positive statistical association to clinical status defined as chronic phase, chronic phase with less than 6 months to blast crisis, accelerated phase and blast crisis. This was in contrast to earlier reports which indicated that patients with breakpoint at 3' site were at a higher biologic risk for entering blast crisis.

Spontaneous cell-mediated cytolysis by peripheral blood cells obtained from whole-body chronically irradiated beagle dogs.

The level of natural killer (NK) activity of continuously gamma-irradiated (whole body) beagle dogs and their nonirradiated controls was studied. For analytical purposes, irradiated dogs were segregated into groups according to their clinical status: clinically normal, hypocellular, or with acute non-lymphocytic leukemia. Since unirradiated control animals exhibited a wide range of NK responses, the data from each irradiated animal were compared to its own age-matched or litter-matched unirradiated control. Of the eight clinically normal irradiated dogs (median = 146% activity of control) only one animal had a NK activity lower than that of its control. The hypocellular group (n = 5, median = 21.8% of control) and the leukemic group (n = 4, median = 52.5% of control) each contained one responder with higher activity than its control. The difference between the percentage of control of the clinically normal and clinically abnormal dogs was found to be significant (P less than 0.05). There is a negative correlation between the NK results obtained and the total accumulated dose of radiation at the time of sampling (correlation coefficient = -0.739, P less than 0.01), suggesting a radiation effect upon natural killer activity, which is evidence by enhancement at lower doses and depression at higher doses of irradiation.

Effect of continuous, whole-body gamma irradiation upon canine lymphohematopoietic (CFU-GM, CFU-L) progenitors and a possible hematopoietic regulatory population.

Clonogenic assays for granulocytes-macrophages (CFU-GM) in bone marrow and for T lymphocytes (CFU-L) in peripheral blood were performed on dogs continuously exposed to 60Co irradiation (0.02, 0.04, or 0.11 Gy/day). When decreased numbers of CFU-GM were observed they correlated well with the clinical status of the dogs but were not generally associated with increasing cumulative doses of absorbed irradiation. In clinically normal, irradiated animals, decreased CFU-GM values and myeloid-erythroid ratios were observed, suggesting that chronic irradiation may affect the granulocytic series well before decreased peripheral blood values are seen. In hypocellular dogs the number of CFU-GM were significantly decreased compared to values obtained from control or clinically normal irradiated dogs, while virtually no CFU-GM were observed in the leukemic dogs. Only the CFU-GM values of the hypocellular group showed an association, e.g., a suggestion of an abortive regenerative effort, with increasing absorbed dose. Proliferative capacity of T lymphocytes (CFU-L) was not affected by either increasing absorbed irradiation or the presence of leukemia. D0 values were determined on marrow fibroblastic cells to ascertain whether a radioresistant subpopulation of stromal elements would result from continuous in vivo irradiation. No correlation was found between absorbed dose and increased D0 values. However, seven of eight dogs which developed acute nonlymphocytic leukemia displayed marrow fibroblastic cells with elevated D0 values. These radioresistant marrow fibroblastic cells were assayed for their ability to support normal granulopoiesis and found to be not significantly different from control fibroblasts.

The influence of fibroblast-like cells derived from canine fetal hematopoietic tissues on the regulation of lymphohematopoiesis.

Media conditioned by fibroblast-like cells derived from organs active in fetal lymphohematopoiesis were studied for their effects on adult granulocyte/macrophage colony-forming units (CFU-GM). Fibroblasts from fetal liver produced a factor stimulatory for CFU-GM, whereas fibroblasts from fetal marrow produced a factor inhibitory for CFU-GM which was not completely relieved by adding indomethacin to the assay. Our studies indicated that neither fetal marrow nor fetal liver produced factors affecting lymphocyte colony-forming units (CFU-L). Cell-cell interactions between fibroblast-like cells derived from fetal liver or marrow and normal adult CFU-GM were also studied. We observed that fibroblasts derived from both fetal and adult marrow inhibited colony formation, whereas inhibition in the presence of fetal liver fibroblasts was minimal. Loss of inhibitory activity by a liver fibroblast cell line over repeated passages was seen. Differential analysis of colonies formed above an adherent layer of fetal marrow fibroblasts suggested that these fibroblasts suppress myeloid/macrophage differentiation to a far greater degree than did adult marrow fibroblasts. A role in the regulation of fetal lymphohematopoiesis may be played by stromal fibroblasts.

Characterization of canine fetal lymphohematopoiesis: studies of CFUGM, CFUL, and CFUF.

We assayed the colony forming units for granulocyte-macrophages (CFUGM), T-lymphocytes (CFUL) and fibroblasts (CFUF) in the blood, bone marrow, liver and spleen of the canine at 45 and 55 days of gestation and 4 and 30 days post partum. As the number of CFUGM per 5 x 10(5) cells increased in the fetal liver, the number of CFUGM increased in circulating blood, whereas when the number of CFUGM decreased in liver and blood, CFUGM increased in both bone marrow and spleen. This suggests that CFUGM are produced in the liver, are released into the circulation and then transported to the spleen and bone marrow. CFUF studies showed that canine fetal bone marrow and spleen are active sites of fibroblast proliferation, whereas the liver is not. Morphologic examination of colonies derived from concanavalin-A stimulated progenitors ("CFUL") demonstrated that these colonies from fetal tissues and adult bone marrow were not exclusively lymphoid but were also made up of significant numbers of precursors of the myeloid and monocytic series. Lymphocyte stimulation tests (LST) showed the presence of a large population of mitogen-independent dividing cells, suggesting that fetal lymphohematopoiesis may be at least partially under the influence of factors other than those of adult cells.

A "whole-blood" technique for the quantitation of canine "T-lymphocyte" progenitors using a semisolid culture system.

A whole blood technique is described for the growth of concanavalin A (Con A) stimulated canine lymphocyte colonies in semisolid medium. By eliminating the routine Ficoll-Paque (F-P) gradient lymphocyte isolation, this method avoids potential problems of growth modulation due to elimination of non-lymphoid accessory cells and the influences on colony formation associated with the selective effects of F-P on lymphocyte subpopulations. Thus, the technique more closely approximates the in vivo milieu. The whole-blood method also produces higher cloning efficiencies than methods using gradient isolation of lymphocytes. Studies over a wide range of blood concentration produced a linear response of in vitro colony formation although extrapolation of the cell-dose colony-response curve did not intersect zero. Mitogen titration data indicates that a relatively large dose of Con A is required for whole blood colony formation compared to the standard F-P method. The colonies ultrastructurally were composed of lymphoblastic and lymphocytic elements which were negative for non-specific esterase activity. Characterization of cells retrieved from the colonies using rosetting techniques indicates a high percentage of the colony cells relative to canine peripheral blood cells form rosettes with human erythrocytes.

The formation of human lymphocyte colonies in semisolid cultures in response to allogeneic mixed-lymphocyte stimulation.

A technique is described for the growth of human lymphocyte colonies in semisolid culture systems in response to allogeneic lymphocyte stimulation. Colonies did not form to any major extent using autologous lymphocyte stimulation. Both one-way and two-way mixed-lymphocyte reactions were investigated. Ultrastructurally, such colonies are composed of cells with lymphoblastic and lymphocytic morphology. The majority of the lymphoid elements composing the colonies were T-cells based on their ability to rosette with sheep red blood cells. Our studies suggest that the colonies are clonogenic in origin and therefore the technique offers the potential for isolation of specific clones, or subpopulations of lymphocytes involved in allogeneic reactions and characterization of their function. Studies directly comparing the stimulation indices achieved with standard mixed lymphocyte cultures utilizing 3HTdr-incorporation to the colony-forming assay indicate that the cloning technique produces higher stimulation indices for allogeneic/autologous reactions and produces less autologous (background) response than the 3HTdr incorporation technique. In addition to lymphocyte colonies, we also observed colonies of surface-adherent populations of macrophages, including multinucleated giant cells. Thus, the technique appears to provide a new and potentially more sensitive method for the study of transplantation immunology and cell-mediated immunity in humans.