Natural killer cell-mediated lysis of autologous cells modified by gene therapy.

This study investigated the role of natural killer (NK) cells as effectors of an immune response against autologous cells modified by gene therapy. T lymphocytes were transduced with LXSN, a retroviral vector adopted for human gene therapy that carries the selectable marker gene neo, and the autologous NK response was evaluated. We found that (i) infection with LXSN makes cells susceptible to autologous NK cell-mediated lysis; (ii) expression of the neo gene is responsible for conferring susceptibility to lysis; (iii) lysis of neo-expressing cells is clonally distributed and mediated only by NK clones that exhibit human histocompatibility leukocyte antigen (HLA)-Bw4 specificity and bear KIR3DL1, a Bw4-specific NK inhibitory receptor; and (iv) the targets are cells from HLA-Bw4(+) individuals. Finally, neo peptides anchoring to the Bw4 allele HLA-B27 interfered with KIR3DL1-mediated recognition of HLA-B27, i.e., they triggered NK lysis. Moreover, neo gene mutations preventing translation of two of the four potentially nonprotective peptides reduced KIR3DL1(+) NK clone-mediated autologous lysis. Thus, individuals expressing Bw4 alleles possess an NK repertoire with the potential to eliminate autologous cells modified by gene therapy. By demonstrating that NK cells can selectively detect the expression of heterologous genes, these observations provide a general model of the NK cell-mediated control of viral infections.

High frequency of clonal immunoglobulin or T cell receptor gene rearrangements in acute myelogenous leukemia expressing terminal deoxyribonucleotidyltransferase.

Ig and T cell receptor rearrangements have been used as irreversible markers of lineage and clonality in the study of B- and T-lymphoid populations. We have addressed the issue of lymphoid lineage specificity of these rearrangements by analyzing a panel of 25 TdT- acute myelogenous leukemias, 13 TdT+ AMLs, and 4 TdT+ undifferentiated leukemias. We report that while gene rearrangements represent extremely rare events in classical TdT- AML (less than 8%), rearrangements of either the Ig or T beta locus or both were detectable in the majority of the TdT+ AMLs (greater than 60%), and rearrangements of both loci were detectable in all of the TdT+ undifferentiated leukemias. These data demonstrate a significant association between TdT expression and Ig or T beta gene rearrangements even outside the lymphoid lineage, further supporting a role for TdT in Ig and T cell receptor gene assembly. These data also indicate that a coordinated program of lymphoid gene expression involving TdT-CD7-expression and Ig/T beta rearrangements can be activated before myeloid commitment. Whether the activation of this program represents a normal, albeit rare, event in early myelopoiesis or a transformation-related event present only in leukemic cells remains to be determined.

Down-regulation of LFA-1 adhesion receptors by C-myc oncogene in human B lymphoblastoid cells.

The function of the c-myc gene and its role in tumorigenesis are poorly understood. In order to elucidate the role of c-myc oncogene activation in B cell malignancy, the phenotypic changes caused by the expression of c-myc oncogenes in human B lymphoblastoid cells immortalized by Epstein-Barr virus were analyzed. C-myc oncogenes caused the down-regulation of lymphocyte function-associated antigen-1 (LFA-1) adhesion molecules (alpha L/beta 2 integrin) and loss of homotypic B cell adhesion in vitro. Down-regulation of LFA-1 occurred by (i) posttranscriptional modulation of LFA-1 alpha L-chain RNA soon after acute c-myc induction, and (ii) transcriptional modulation in cells that chronically express c-myc oncogenes. Analogous reductions in LFA-1 expression were detectable in Burkitt lymphoma cells carrying activated c-myc oncogenes. Since LFA-1 is involved in B cell adhesion to cytotoxic T cells, natural killer cells, and vascular endothelium, these results imply functions for c-myc in normal B cell development and lymphomagenesis.

The PML/RARalpha fusion protein inhibits tumor necrosis factor-alpha-induced apoptosis in U937 cells and acute promyelocytic leukemia blasts.

We investigated the effect of the acute promyelocytic leukemia (APL) specific PML/RARalpha fusion protein on the sensitivity to TNF-alpha-mediated apoptosis. The U937 leukemia cell line was transduced with PML/RARalpha cDNA. PML/RARalpha expression caused a markedly reduced sensitivity to TNF-alpha, even if apoptosis was triggered by agonistic antibodies to TNF-alpha receptors I and II (TNF-alphaRI, II). PML/RARalpha induced a 10-20-fold decrease of the TNF-alpha-binding capacity via downmodulation of both TNF-alphaRI and TNF-alphaRII: this may mediate at least in part the reduced sensitivity to TNF-alpha. Furthermore, the fusion protein did not modify Fas expression (CD95) or sensitivity to Fas-mediated apoptosis. The pathophysiological significance of these findings is supported by two series of observations. (a) Fresh APL blasts exhibit no TNF-alpha binding and are resistant to TNF-alpha-mediated apoptosis. Conversely, normal myeloblasts-promyelocytes show marked TNF-alphaR expression and are moderately sensitive to TNF-alpha-mediated cytotoxicity. Similarly, blasts from other types of acute myeloid leukemia (AML M1, M2, and M4 FAB types) show an elevated TNF-alpha binding. (b) The NB4 APL cell line, which is PML/RARalpha+, shows low TNF-alphaR expression capacity and is resistant to TNF-alpha-triggered apoptosis; conversely a PML/RARalpha- NB4 subclone (NB4.306) exhibits detectable TNF-alpha-binding capacity and is sensitive to TNF-alpha-mediated cytotoxicity. These studies indicate that the PML/RARalpha fusion protein protects against TNF-alpha-induced apoptosis, at least in part via downmodulation of TNF-alphaRI/II: this phenomenon may play a significant role in APL, which is characterized by prolonged survival of leukemic blasts.

Opposite effects of the acute promyelocytic leukemia PML-retinoic acid receptor alpha (RAR alpha) and PLZF-RAR alpha fusion proteins on retinoic acid signalling.

Fusion proteins involving the retinoic acid receptor alpha (RAR alpha) and the PML or PLZF nuclear protein are the genetic markers of acute promyelocytic leukemias (APLs). APLs with the PML-RAR alpha or the PLZF-RAR alpha fusion protein are phenotypically indistinguishable except that they differ in their sensitivity to retinoic acid (RA)-induced differentiation: PML-RAR alpha blasts are sensitive to RA and patients enter disease remission after RA treatment, while patients with PLZF-RAR alpha do not. We here report that (i) like PML-RAR alpha expression, PLZF-RAR alpha expression blocks terminal differentiation of hematopoietic precursor cell lines (U937 and HL-60) in response to different stimuli (vitamin D3, transforming growth factor beta1, and dimethyl sulfoxide); (ii) PML-RAR alpha, but not PLZF-RAR alpha, increases RA sensitivity of hematopoietic precursor cells and restores RA sensitivity of RA-resistant hematopoietic cells; (iii) PML-RAR alpha and PLZF-RAR alpha have similar RA binding affinities; and (iv) PML-RAR alpha enhances the RA response of RA target genes (those for RAR beta, RAR gamma, and transglutaminase type II [TGase]) in vivo, while PLZF-RAR alpha expression has either no effect (RAR beta) or an inhibitory activity (RAR gamma and type II TGase). These data demonstrate that PML-RAR alpha and PLZF-RAR alpha have similar (inhibitory) effects on RA-independent differentiation and opposite (stimulatory or inhibitory) effects on RA-dependent differentiation and that they behave in vivo as RA-dependent enhancers or inhibitors of RA-responsive genes, respectively. Their different activities on the RA signalling pathway might underlie the different responses of PML-RAR alpha and PLZF-RAR alpha APLs to RA treatment. The PLZF-RAR alpha fusion protein contains an approximately 120-amino-acid N-terminal motif (called the POZ domain), which is also found in a variety of zinc finger proteins and a group of poxvirus proteins and which mediates protein-protein interactions. Deletion of the PLZF POZ domain partially abrogated the inhibitory effect of PLZF-RAR alpha on RA-induced differentiation and on RA-mediated type II TGase up-regulation, suggesting that POZ-mediated protein interactions might be responsible for the inhibitory transcriptional activities of PLZF-RAR alpha.

The promyelocytic leukemia gene product (PML) forms stable complexes with the retinoblastoma protein.

PML is a nuclear protein with growth-suppressive properties originally identified in the context of the PML-retinoic acid receptor alpha (RAR alpha) fusion protein of acute promyelocytic leukemia. PML localizes within distinct nuclear structures, called nuclear bodies, which are disrupted by the expression of PML-RAR alpha. We report that PML colocalizes with the nonphosphorylated fraction of the retinoblastoma protein (pRB) within nuclear bodies and that pRB is delocalized by PML-RAR alpha expression. Both PML and PML-RAR alpha form complexes with the nonphosphorylated form of pRB in vivo, and they interact with the pocket region of pRB. The regions of PML and PML-RAR alpha involved in pRB binding differ; in fact, the B boxes and the C-terminal region of PML, the latter of which is not present in PML-RAR alpha, are essential for the formation of stable complexes with pRB. Functionally, PML abolishes activation of glucocorticoid receptor-regulated transcription by pRB, whereas PML-RAR alpha further increases it. Our results suggest that PML may be part of transcription-regulatory complexes and that the oncogenic potential of the PML-RAR alpha protein may derive from the alteration of PML-regulated transcription.

N-(4-hydroxyphenyl)retinamide induces apoptosis of malignant hemopoietic cell lines including those unresponsive to retinoic acid.

N-(4-Hydroxyphenyl)retinamide (HPR) is a synthetic retinoid of particular clinical interest in cancer chemoprevention. We have examined the in vitro effects of HPR on lymphoid and myeloid malignant cell lines and found that at concentrations between 10(-5) and 3 x 10(-7) M it induces a dose-dependent growth inhibition (the peak plasma concentration in patients treated with HPR is 1 to 2 x 10(-6) M). The antiproliferative effect of HPR was, in all cell lines except K422, more potent than that induced by an equimolar dose of all-trans retinoic acid (RA). Also, this effect was irreversible on HL60 and DoHH2 cells that had been exposed to HPR (3 x 10(-6) M) for 24 h, but reversible on Raji and DHL4 exposed to the retinoid for 48 and 72 h, respectively. Time-course growth analysis showed that HPR at 3 x 10(-6) M or below induces a rapid fall of thymidine uptake and viability (> 90%), whereas between 10(-6) and 3 x 10(-7) M exhibits cytostatic effects. Interestingly, the RA-resistant HL-60R and NB306 cells, characterized by a point mutation in the retinoic acid receptor (RAR) and by the loss of the pml/RAR protein, respectively, were, like the parental RA-inducible HL-60 and NB4 cell lines, fully responsive to HPR, thereby suggesting that HPR and RA could act through different receptors or pathways. DNA flow-cytofluorimetric analysis revealed that HPR does not block cells in a specific phase of the cell cycle but triggers programmed cell death or apoptosis. This phenomenon was evidenced both by the visualization, on gel electrophoresis, of fragmented DNA, and by the "in cell" enzymatic labeling of DNA breaks with fluorescent dUTP. With the latter method, apoptotic cells become detectable by 6 h following exposure to 3 x 10(-6) M HPR. Ultrastructural examination of HPR-treated samples showed cells with chromatin compaction and cytoplasm condensation, characteristic of apoptotic cells. In conclusion, our study demonstrates that HPR suppresses malignant cell growth and induces apoptosis at pharmacologically relevant doses. The differential responsiveness by a number of cell lines, especially HL-60R and NB306, to HPR and RA indicates that these compounds may act through different receptors. The clinical use of HPR, particularly in retinoic acid-unresponsive acute promyelocytic leukemia patients, is suggested.

High-efficiency gene transfer and selection of human hematopoietic progenitor cells with a hybrid EBV/retroviral vector expressing the green fluorescence protein.

We report a retroviral expression vector (PINCO) that allows high-efficiency gene transfer and selection of hemopoietic progenitor cells (HPCs). The main characteristics of this vector are the presence outside the two long terminal repeats of the EBV origin of replication and the EBNA-1 gene and the presence in the retrovirus of the cDNA that encodes for the enhanced green fluorescence protein (GFP), controlled by a cytomegalovirus promoter. Transient transfection of PINCO in Phoenix packaging cells results in episomal propagation of the plasmid and generates viral titers as high as 10(7) colony-forming units/ml. Infection of established cell lines with the PINCO retrovirus yields more than 95% GFP-expressing cells. GFP expression remains stable for months in infected cell cultures and can easily be monitored by fluorescent microscopy or fluorescence-activated cell-sorting (FACS) analysis of living cells. The PINCO vector allows efficient expression of a second gene (thymidine kinase, Shc, and PML), and there is strict correlation between GFP and second gene expression levels in the infected cells. PINCO was used to infect human HPCs; infection efficiency was about 50%. GFP-positive cells can be FACS sorted to yield a homogeneous population of infected cells. FACS-sorted GFP-positive HPC cells have, with respect to unfractionated HPC cells, the same frequency of long-term culture initiating cells and an identical capacity to undergo multilineage and unilineage differentiation. The entire gene transfer procedure, from the transfection of the packaging cell line to the infection of target cells, requires less than a week. The high viral titer and the easy obtainment of homogeneously infected cell populations without drug selection procedures make PINCO an ideal vector for gene transfer of human primary hemopoietic cells.

Promyelocytic leukemia-specific PML-retinoic acid alpha receptor fusion protein interferes with erythroid differentiation of human erythroleukemia K562 cells.

Acute promyelocytic leukemia (APL) is characterized by a t(15;17) chromosomal translocation with breakpoints within the retinoic acid alpha receptor (RAR alpha) gene on 17 and the PML gene, which encodes a putative transcription factor, on 15. A PML-RAR alpha fusion protein is formed as a consequence of the translocation. We show here that expression of the PML-RAR alpha protein in K562 erythroleukemia cells results in a reduced expression of erythroid differentiation markers and a reduced sensitivity to the erythroid differentiative action of heme. Overexpression of RAR alpha, but not of PML, elicited a similar inhibition of K562 erythroid differentiation. These findings indicate that overexpression of either RAR alpha or PML/RAR alpha interferes with erythroid differentiation and support the hypothesis that RAR alpha is involved in the regulation of normal hematopoiesis and alteration of the RAR alpha signaling by PML/RAR alpha is implicated in the promyelocytic leukemogenesis.

Characterization of the PML-RAR alpha chimeric product of the acute promyelocytic leukemia-specific t(15;17) translocation.

The acute promyelocytic leukemia 15;17 chromosomal translocation fuses the PML gene to the RAR alpha locus. The resulting chimeric gene encodes for a putative PML-RAR alpha fusion protein. PML is a putative transcriptional factor and RAR alpha is one of the nuclear retinoic acid receptors through which retinoic acid regulates gene expression. In this study, we investigated the retinoid binding and biochemical properties of the PML-RAR alpha protein by size exclusion high-performance liquid chromatography and immunoblot analysis and compared them with those of normal RAR alpha. The introduction of the expression vector PSG5/PML-RAR alpha into COS-1 cells led to high levels of expression of the PML-RAR alpha fusion protein. This protein was primarily localized in the nucleus and bound retinoids with the same affinity and specificity as the wild type RAR alpha receptor. The PML-RAR alpha fusion protein, but not the RAR alpha, was found in high molecular weight complexes with either itself or other nuclear factors. In the acute promyelocytic leukemia-derived cell line NB4, which contains the t(15;17) chromosomal marker, the PML-RAR alpha product was also found as a high molecular complex. The interaction of the PML-RAR alpha with itself or with other nuclear proteins may be important in understanding the role of the PML-RAR alpha fusion protein in promyelocytic leukemogenesis.

The localization of the HRX/ALL1 protein to specific nuclear subdomains is altered by fusion with its eps15 translocation partner.

Translocations involving the HRX/ALL1 locus at chromosomal region 11q23 are among the most frequent cytogenetic abnormalities in acute leukemias. 11q23 translocations involve different chromosome partners and lead to the formation of HRX/ALL1 fusion proteins. The HRX/ALL1 protein is a putative transcription factor that has been implicated in developmental regulation in mammals. We report here the cellular localization of the HRX/ALL1 protein as well as that of the HRX/ALL1-eps15 fusion protein, the result of the t(1;11) (p32-q23) translocation of acute myeloid leukemias. The HRX/ALL1 protein was localized to both the cytoplasm and the nucleus. The nuclear pattern was characterized by diffuse staining, perinuclear accumulation, and localization within nuclear bodies of variable size, morphology, and number. The HRX/ALL1-eps15 localized exclusively to the nucleus within bodies that were smaller and more numerous than the HRX/ALL1 nuclear bodies. HRX/ALL1 fusion with an unknown partner in leukemia blasts with 11q23 abnormalities had similar morphological features. Thus, the fusion with eps15 alters the cellular compartmentalization of HRX/ALL1, providing a putative mechanism for activation of HRX/ALL1 by 11q23 abnormalities.

PML/RAR alpha+ U937 mutant and NB4 cell lines: retinoic acid restores the monocytic differentiation response to vitamin D3.

We have analyzed the differentiation program of a U937 promonocytic leukemia clone transduced with the acute promyelocytic leukemia specific PML/RAR alpha fusion gene, the expression of which is under the control of the inducible metallothionine (MT) I promoter (MTPR9 clone). MTPR9 cells treated with Zn2+ hence exhibit levels of PML-RAR alpha protein as high as fresh acute promyelocytic leukemia blasts. In the absence of Zn2+, i.e., upon low level PML/RAR alpha expression, 1,25-dihydroxyvitamin D3 (D3) and particularly D3 plus transforming growth factor beta 1 (TGF-beta 1) induced terminal differentiation of MTPR9 cells (as observed in "wild-type" U937 cells), on the basis of morphology, membrane antigen pattern, and functional criteria. Conversely, in the presence of Zn2+, D3 and D3 plus TGF-beta 1 failed to induce terminal differentiation, as evaluated by the above parameters. Interestingly, retinoic acid (RA) treatment suppresses the differentiation blockade induced by high level PML-RAR alpha protein; indeed, Zn(2+)-treated MTPR9 cells incubated with RA plus D3 exhibited significant terminal monocytic maturation, comparable to that of cells treated with D3 alone or combined with RA in absence of Zn2+. Similar observations were made in NB4, a PML-RAR+ human acute leukemic line. As expected RA treatment of NB4 cells causes granulocytic differentiation. Interestingly, the cell line is only scarcely induced to mature monocytic cells by D3 or D3 plus TGF-beta 1 treatment, whereas it is effectively induced to monocytic maturation by combined treatment with D3 and RA. Accordingly, the rate of NB4 cell proliferation is only slightly affected by D3 or D3 plus TGF-beta 1 treatment, mildly inhibited by RA, and markedly decreased by D3 plus RA. These results indicate that in both U937 and NB4 cells high level PML/RAR alpha expression inhibits the monocytic terminal differentiation program triggered by D3 or D3 plus TGF-beta 1, whereas RA treatment effectively antagonizes this inhibitory PML-RAR alpha action and restores the D3 differentiative effect.

PML is required for telomere stability in non-neoplastic human cells.

Telomeres interact with numerous proteins, including components of the shelterin complex, whose alteration, similarly to proliferation-induced telomere shortening, initiates cellular senescence. In tumors, telomere length is maintained by Telomerase activity or by the Alternative Lengthening of Telomeres mechanism, whose hallmark is the telomeric localization of the promyelocytic leukemia (PML) protein. Whether PML contributes to telomeres maintenance in normal cells is unknown. We show that in normal human fibroblasts the PML protein associates with few telomeres, preferentially when they are damaged. Proliferation-induced telomere attrition or their damage due to alteration of the shelterin complex enhances the telomeric localization of PML, which is increased in human T-lymphocytes derived from patients genetically deficient in telomerase. In normal fibroblasts, PML depletion induces telomere damage, nuclear and chromosomal abnormalities, and senescence. Expression of the leukemia protein PML/RARα in hematopoietic progenitors displaces PML from telomeres and induces telomere shortening in the bone marrow of pre-leukemic mice. Our work provides a novel view of the physiologic function of PML, which participates in telomeres surveillance in normal cells. Our data further imply that a diminished PML function may contribute to cell senescence, genomic instability, and tumorigenesis.

Cooperation between the RING + B1-B2 and coiled-coil domains of PML is necessary for its effects on cell survival.

PML/RARalpha is the abnormal protein product of the Acute Promyelocytic Leukemia-specific 15;17 translocation. Both the PML and RARalpha components are required for the PML/RARalpha biological activities, namely its capacity to block differentiation and to increase survival of haematopoietic precursors. The physiological role of PML and its contribution to the function of the fusion protein are unknown. PML localizes to the cytoplasm and within specific nuclear bodies (NBs). In vitro, overexpression of PML correlates with suppression of cell transformation. The PML aminoterminal portion retained within the PML/RARalpha protein contains the RING finger, two newly defined cystein/histidine-rich motifs called B-boxes (B1 and B2) and a coiled-coil region. We report here that PML has a growth suppressive activity in all the cell lines tested, regardless of their transformed phenotype, and that the cellular basis for the PML growth suppression is induction of apoptotic cell death. Analysis of various nuclear and cytoplasmic PML isoforms showed that the PML growth suppressive activity correlates with its nuclear localization. Analysis of the localization and growth suppressive activity demonstrated that: (i) the Ring + B1-B2 and coiled-coil regions are both indispensable and sufficient to target PML to the NBs; (ii) individual deletions of the various PML domains have no effect on its growth suppressor activity; (iii) the Ring + B1-B2 region exerts a partial growth suppressor activity but its fusion with the coiled-coil region is sufficient to recapitulate the suppressive function of wild type PML. These results indicate that PML is involved in cell survival regulation and that the PML component of the fusion protein (Ring + B1-B2 and coiled-coil regions) retains intact biological activity, thereby suggesting that the effects of PML/RARalpha on survival derive from the activation of the incorporated PML sequence.

Formation of PML/RAR alpha high molecular weight nuclear complexes through the PML coiled-coil region is essential for the PML/RAR alpha-mediated retinoic acid response.

Retinoic Acid (RA) treatment induces disease remission of Acute Promyelocytic Leukaemia (APL) patients by triggering terminal differentiation of neoplastic cells. RA-sensitivity in APL is mediated by its oncogenic protein, which results from the recombination of the PML and the RA receptor alpha (RAR alpha) genes (PML/RAR alpha fusion protein). Ectopic expression of PML/RAR alpha into haemopoietic cell lines results in increased response to RA-induced differentiation. By structure-function analysis of PML/RAR alpha-mediated RA-differentiation, we demonstrated that fusion of PML and RAR alpha sequences and integrity of the PML dimerization domain and of the RAR alpha DNA binding region are required for the effect of PML/RAR alpha on RA-differentiation. Indeed, direct fusion of the PML dimerization domain to the N- or C-terminal extremities of RAR alpha retained full biological activity. All the biologically active PML/RAR alpha mutants formed high molecular weight complexes in vivo. Functional analysis of mutations within the PML dimerization domain revealed that the capacity to form PML/RAR alpha homodimers, but not PML/RAR alpha-PML heterodimers, correlated with the RA-response. These results suggest that targeting of RAR alpha sequences by the PML dimerization domain and formation of nuclear PML/RAR alpha homodimeric complexes are crucial for the ability of PML/RAR alpha to mediate RA-response.

Structure and origin of the acute promyelocytic leukemia myl/RAR alpha cDNA and characterization of its retinoid-binding and transactivation properties.

Acute promyelocytic leukemia (APL) is characterized by the 15;17 chromosomal translocation. Cloning experiments have established that the chromosome 17 breakpoint maps to the RAR alpha and the 15 to the myl locus. The resulting chimeric gene is transcribed as a myl/RAR alpha fusion mRNA. By isolating both normal myl and APL myl/RAR alpha cDNAs, we showed that the myl/RAR alpha mRNA encodes for a putative fusion protein with a molecular weight of about 103 kDa, which is made up of 530 amino acids derived from the myl N-terminus and 402 amino acids originating from the RAR alpha C-terminus. The protein includes the RAR alpha DNA and retinoid-binding regions but lacks the A portion of the N-terminal region (A/B region) which is thought to contain one of the RAR alpha transactivation domains. The myl/RAR alpha protein acted as a retinoid-inducible transcription factor with both ligand-independent repressor and ligand-dependent activator functions in transactivation experiments of a retinoic acid-responsive gene. Myl/RAR alpha exerted this dual function three times more effectively than RAR alpha and had about 10-fold greater affinity for RA than RAR alpha. Comparison of myl/RAR alpha genomic and cDNA sequences from the same case demonstrated that both chromosome 15 and 17 breakpoints occurred within introns and the myl and RAR alpha sequences are spliced in the same polyadenylated RNA.

Characterization of human and mouse c-fes cDNA clones and identification of the 5' end of the gene.

cDNA clones of the human c-fes mRNA were isolated. Nucleotide analysis showed that c-fes mRNA contains a 2514 nucleotide open reading frame, which could encode for a 93 kDa protein, and both 5' and 3' v-fes nonhomologous sequences. Primer extension experiments confirmed that the longest isolated cDNAs are about the same length as the entire human c-fes mRNA. Sequence comparison between human c-fes cDNA and the corresponding genomic regions identified a 5' viral-non homologous exon (exon 1) located 491 bp upstream from the first v-fes homologous exon. The genomic region surrounding c-fes exon 1 contains a CpG island and acts as a promoter in vitro. Analysis of the 5' end of mouse c-fes cDNA suggested that the 5' human and mouse gene structure are similar.

Alternative splicing of PML transcripts predicts coexpression of several carboxy-terminally different protein isoforms.

The acute promyelocytic leukaemia (APL)-specific chromosome 15;17 translocation leads to the fusion of a newly identified putative transcription factor, PML, and the retinoic acid receptor alpha. We have characterized the structure of the PML genomic locus and preliminarily characterized its expression pattern. The PML locus spans a minimum of 35 kb and is subdivided into nine exons. The putative PML DNA binding site is encoded by exons 2 and 3. We isolated a large number of alternatively spliced PML transcripts that encode numerous PML isoforms. Two groups of isoforms were identified that differed either in their C-terminal region or in the length of their central region, but retained the putative DNA-binding and dimerization domains. RNAase protection experiments revealed that the different PML isoforms are equally expressed in established cell lines of different histological origin.

Expression and role of PML gene in normal adult hematopoiesis: functional interaction between PML and Rb proteins in erythropoiesis.

The expression of the PML gene was investigated in purified early hematopoietic progenitor cells (HPCs) induced to unilineage erythroid or granulocytic differentiation. PML mRNA and protein, while barely detectable in quiescent HPCs, are consistently induced by growth factor stimulation through the erythroid or granulocytic lineage. Thereafter, PML is downmodulated in late granulocytic maturation, whereas it is sustainably expressed through the erythroid pathway. In functional studies, PML expression was inhibited by addition of antisense oligomers targeting PML mRNA (alpha-PML). Interestingly, early treatment (day 0 HPCs) with alpha-PML reduced the number of both erythroid and granulocytic colonies, whereas late treatment (day 5 culture) reduced erythroid, but not granulocytic, clonogenesis. These findings suggest that PML is required for early hematopoiesis and erythroid, but not granulocytic maturation. The pattern of PML expression in normal hematopoiesis mimics that of retinoblastoma pRb 105. Combined treatment of HPCs with alpha-PML and alpha-Rb oligomers inhibited both PML and Rb protein expression and completely blocked erythroid colony development. Furthermore, PML and pRb 105 were co-immunoprecipitated in cellular lysates derived from erythroid precursors indicating that this functional interaction may have a biochemical basis. These results suggest a key functional role of PML in early hematopoiesis and late erythropoiesis: the latter phenomenon may be related to the molecular and functional interaction of PML with pRb 105.

The acute promyelocytic leukaemia specific PML and PLZF proteins localize to adjacent and functionally distinct nuclear bodies.

Acute promyelocytic leukaemia is characterized by translocations that involve the retinoic acid receptor alpha (RAR alpha) locus on chromosome 17 and the PML locus on 15 or the PLZF locus on 11. The resulting abnormal translocation products encode for PML/RAR alpha or PLZF/RAR alpha fusion proteins. There is increasing experimental evidence that the APL-specific fusion proteins have similar biologic activities on differentiation and survival and that both components of the fusion proteins (PML or PLZF and RAR alpha) are indispensable for these biological activities. The physiologic function of PML or PLZF or whether PML and PLZF contribute common structural or functional features to the corresponding fusion proteins is not known. We report here immunofluorescence studies on the cellular localization of PLZF and PLZF/RAR alpha and compare it with the localization of PML and PML/RAR alpha. PLZF localizes to nuclear domains of 0.3-0.5 microns, approximately 14 per cell in the KG1 myeloid cell line. These PLZF-bodies are morphologically similar to the domains reported for PML (PML-NBs). There is tight spatial relationship between about 30% of PLZ-NBs and PML-NBs: they partially overlap. However, PML and PLZF do not form soluble complexes in vivo. PLZF- and PML-NBs are functionally distinct. Adenovirus E4-ORF3 protein expression alters the structure of the PML-NBs and interferon increases the number of PML-NBs and neither has any effect on PLZF NBs. The localization of PLZF/RAR alpha is different to that of PLZF and RAR alpha. The nuclear distribution pattern of PLZF/RAR alpha is one of hundreds of small dots (microspeckles) less than 0.1 micron. Expression of PLZF/RAR alpha did not provoke disruption of the PML-NBs. Co-expression of PML/RAR alpha and PLZF/RAR alpha in U937 cells revealed apparent colocalization. Overall the results suggest that the PML- and PLZF-NBs are distinct functional nuclear domains, but that they may share common regulatory pathways and/or targeting sequences, as revealed by the common localization of their corresponding fusion proteins.