Transcriptional activation of the cyclin-dependent kinase inhibitor p21 by PML/RARalpha.

Acute promyelocytic leukemia (APL) is a result of clonal expansion of hematopoietic precursors blocked at the promyelocytic stage and is associated with a t(15;17) chromosomal translocation and the expression of the PML/RARalpha fusion protein. Treatment of APL cells with retinoic acid (RA) leads to complete remission by inducing growth arrest and differentiation of these cells into granulocytes. The cyclin-dependent kinase inhibitor p21WAF1/CIP1 may be involved in terminal differentiation associated growth arrest. We showed in this study that PML/RARalpha increased the transcription of p21WAF1/CIP1 gene and the activation was further induced by RA treatment. Deletion analysis revealed a region upstream of the p21WAF1/CIP1 promoter that is required for transactivation by PML/RARalpha. Transient transfection of PML/RARalpha in cells increased the endogenous p21WAF1/CIP1 protein levels. These results suggest that the induction of APL cells differentiation by RA may be a result of the activation of p21WAF1/CIP1 by PML/RARalpha.

Leukemia translocation protein PLZF inhibits cell growth and expression of cyclin A.

The PLZF gene was identified by its fusion with the RARalpha locus in a therapy resistant form of acute promyelocytic leukemia (APL) associated with the t(11;17)(q23;q21) translocation. Here we describe PLZF as a negative regulator of cell cycle progression ultimately leading to growth suppression. PLZF can bind and repress the cyclin A2 promoter while expression of cyclin A2 reverts the growth suppressed phenotype of myeloid cells expressing PLZF. In contrast RARalpha-PLZF, a fusion protein generated in t(11;17)(q23;q21)-APL activates cyclin A2 transcription and allows expression of cyclin A in anchorage-deprived NIH3T3 cells. Therefore, cyclin A2 is a candidate target gene for PLZF and inhibition of cyclin A expression may contribute to the growth suppressive properties of PLZF. Deregulation of cyclin A2 by RARalpha-PLZF may represent an oncogenic mechanism of this chimeric protein and contribute to the aggressive clinical phenotype of t(11;17)(q23;q21)-associated APL.

Targeting the PML/RAR alpha translocation product triggers apoptosis in promyelocytic leukemia cells.

The t(15;17) rearrangement found in acute promyelocytic leukemia (APL) yields a fusion transcript, PML/RAR alpha. PML/RAR alpha expression is linked to leukemogenesis and to clinical sensitivity to all-trans retinoic acid (RA). Paradoxically, RA treatment causes transient complete remissions in most t(15;17) APL cases. The precise roles of PML/RAR alpha in triggering leukemia or in causing a maturation block are not yet known. This study explores directly these PML/RAR alpha functions in the growth and differentiation of APL cells using a hammerhead ribozyme to target PML/RAR alpha mRNA in the NB4 APL cell line. When the PML/RAR alpha cleaving but not the non-catalytic control ribozyme is introduced into the NB4 APL cell line, PML/RAR alpha protein expression is reduced. This catalysis signals growth suppression, cytotoxicity, and apoptosis without overcoming the maturation block found in these leukemic cells. These biologic effects depend on the selective pressure used to express the ribozyme from an episomal vector. Introduction of a non-catalytic, control ribozyme into NB4 cells caused no observed phenotype due to anti-sense activities. Expression of the catalytic or non-catalytic ribozymes in control cells lacking PML/RAR alpha mRNA yielded no apparent growth or differentiation effects. Thus, use of a hammerhead ribozyme that targets PML/RAR alpha expression in APL cells reveals the anti-apoptotic function of this translocation product and demonstrates that PML/RAR alpha cleavage is insufficient to overcome the differentiation block observed in these leukemic cells. Taken together, these findings indicate that persistent PML/RAR alpha expression is required to maintain basal leukemic cell growth and point to the therapeutic potential of targeting PML/RAR alpha in APL.

Targeting of PML/RARalpha is lethal to retinoic acid-resistant promyelocytic leukemia cells.

Acute promyelocytic leukemia (APL) cells, containing the t(15;17) rearrangement, express the fusion protein, PML/RARalpha. Clinically, patients respond to all-trans retinoic acid (ATRA) through complete remissions associated with myeloid maturation of leukemic cells. This clinical ATRA response of APL is linked to PML/RARalpha expression. Unfortunately, these remissions are transient and relapsed APL is often ATRA-resistant. The role PML/RARalpha plays in the growth and maturation of these APL cells with acquired ATRA resistance has not been fully explored. This study uses an ATRA-resistant NB4 cell line (NB4-R1) to investigate the contribution of PML/RARalpha expression to ATRA resistance. Targeting of PML/RARalpha in NB4-R1 cells was undertaken using two approaches: homologous recombination and hammerhead ribozyme-mediated cleavage. Reducing PML/RARalpha protein in NB4-R1 cells rendered these cells more sensitive to ATRA. These cells were growth-inhibited in ATRA, apoptosis was induced, and there was no apparent signaling of differentiation. Sequence analysis identified a mutation in the ligand binding domain (LBD) of the RARalpha portion of PML/RARalpha. Results show that these retinoid-resistant NB4 cells require persistent PML/RARalpha expression for leukemic cell growth. Taken together, these findings can account for why these cells do not respond to ATRA and how reduction of PML/RARalpha abrogates the antiapoptotic effect it confers to these leukemic cells.

Common defects of different retinoic acid resistant promyelocytic leukemia cells are persistent telomerase activity and nuclear body disorganization.

The acute promyelocytic leukemia (APL) t(15;17) rearrangement fuses the promyelocytic leukemia (PML) gene to the retinoic acid receptor-alpha (RAR alpha). There is expression of the chimeric transcript, PML/RAR alpha, in these APL cells. These clinical APL cases respond to the differentiation agent all-trans retinoic acid (ATRA) with complete but not durable remissions because ATRA resistance develops. The NB4 APL cell line expresses PML/RAR alpha and responds to the growth inhibitory and differentiation-inducing signals of ATRA. To identify mechanisms responsible for ATRA resistance in APL, ATRA-resistant NB4 cell lines were derived from parental NB4 cells using different strategies. These lines were resistant to the growth inhibition and differentiation effects of ATRA. ATRA-resistant cells were isolated as a de novo resistant line from parental NB4 cells (NB4-R1), following chemical mutagenization and selection in ATRA (NB4-R2), or after chronic selection in ATRA (NB4-R3). Common defects linked to this ATRA resistance were found. When cultured in ATRA, these resistant cells still express PML, RAR alpha, and PML/RAR alpha proteins. Sequence abnormalities were not detected in the RAR alpha DNA binding domains cloned from a representative RA-resistant NB4 line. In ATRA-sensitive but not ATRA-resistant NB4 cells, ATRA down-regulated retinoid X receptor-alpha (RXR alpha) expression, a known marker of ATRA response in parental NB4 cells. Notably, engineered overexpression of RXR alpha in ATRA-sensitive NB4 cells did not block ATRA-mediated growth suppression. ATRA treatment of these resistant NB4 lines did not signal a decline in telomerase activity or reorganization of PML-associated nuclear bodies, but both events occurred in ATRA-sensitive NB4 cells. These ATRA-resistant NB4 lines are not fully differentiation-defective, since monocytic maturation was induced following treatment with phorbol 12-myristate 13-acetate (PMA) and 1,25 dihydroxy vitamin D3 (vitamin D3). Notably, induced monocytic differentiation of these distinct ATRA-resistant APL lines markedly repressed telomerase activity. Thus, this study suggests that persistent telomerase activity and nuclear body disorganization are linked to ATRA resistance in APL.

Interferon augments PML and PML/RAR alpha expression in normal myeloid and acute promyelocytic cells and cooperates with all-trans retinoic acid to induce maturation of a retinoid-resistant promyelocytic cell line.

The PML gene is fused to the retinoic acid receptor alpha gene (RAR alpha) in the acute promyelocytic leukemia (APL) 15; 17 translocation. PML is expressed in diverse tissues and cell lines and localized in the nucleus with a typical speckled pattern. In the bone marrow, it is preferentially expressed in myeloid cells. PML appears to be transcriptionally regulated by class I and II interferons, which raises the possibility that interferons modulate the function and growth and differentiation potential of normal myeloid cells and precursors by activating PML-dependent pathways. Similarly, interferons could act on APL cells, alone or in combination with all-trans retinoic acid (RA), especially if the PML/RAR alpha fusion transcript that results from the t(15; 17) is induced by interferon. We report here that PML is expressed at low levels or not expressed in normal circulating human monocytes, lymphocytes, and polymorphonucleate cells, but is markedly induced by interferon; that PML and PML/RAR alpha expression is augmented by interferon in the NB4 APL cell line, which carries the t(15; 17), and in APL blasts from patients; that interferon inhibits growth and survival of NB4 APL cells in cooperation with RA; that interferons alone have minimal maturation effect on NB4 cells; and, finally, that interferon gamma, but not alpha or beta, induces maturation and growth suppression of NB4 cells with de novo retinoid resistance, and partially restores RA response.

Transgenic expression of PML/RARalpha impairs myelopoiesis.

The translocation found in acute promyelocytic leukemia rearranges the promyelocytic leukemia gene (PML) on chromosome 15 with the retinoic acid receptor alpha (RARalpha) on chromosome 17. This yields a fusion transcript, PML/RARalpha, a transcription factor with reported dominant negative functions in the absence of hormone. Clinical remissions induced with all-trans retinoic acid (RA) treatment in acute promyelocytic leukemia are linked to PML/RARalpha expression in leukemic cells. To evaluate the PML/RARalpha role in myelopoiesis, transgenic mice expressing PML/RARalpha were engineered. A full-length PML/RARalpha cDNA driven by the CD11b promoter was expressed in transgenic mice. Expression was confirmed in the bone marrow with a reverse transcription PCR assay. Basal total white blood cell and granulocyte counts did not appreciably differ between PML/RARalpha transgenic and control mice. Cell sorter analysis of CD11b+ bone marrow cells revealed similar CD11b+ populations in transgenic and control mice. However, in vitro clonal growth assays performed on peripheral blood from transgenic versus control mice revealed a marked reduction of myeloid progenitors, especially in those responding to granulocyte/ macrophage colony-stimulating factor. Granulocyte/macrophage colony-stimulating factor and kit ligand cotreatment did not overcome this inhibition. Impaired myelopoiesis in vivo was shown by stressing these mice with sublethal irradiation. Following irradiation, PML/RARalpha transgenic mice, as compared with controls, more rapidly depressed peripheral white blood cell and granulocyte counts. As expected, nearly all control mice (94.4%) survived irradiation, yet this irradiation was lethal to 45.8% of PML/RARalpha transgenic mice. Lethality was associated with more severe leukopenia in transgenic versus control mice. Retinoic acid treatment of irradiated PML/RARalpha mice enhanced granulocyte recovery. These data suggest that abnormal myelopoiesis due to PML/RARalpha expression is an early event in oncogenic transformation.

Growth suppression of acute promyelocytic leukemia cells having increased expression of the non-rearranged alleles: RAR alpha or PML.

The balanced t(15;17) rearrangement found in acute promyelocytic leukemia (APL) cells fuses PML on chromosome 15 to the retinoic acid receptor alpha (RAR alpha) on chromosome 17. PML/RAR alpha is expressed in APL cells with the non-rearranged alleles, PML and RAR alpha. Clinical remissions induced by all-trans-retinoic acid (RA) treatment of APL patients are linked to expression of PML/RAR apha, a transcription factor with reported dominant negative functions. The roles of PML and RAR alpha in the RA response of APL have not yet been fully explored. This study examines these roles by individually transfecting RAR alpha and PML into NB4 APL cells. NB4 is the sole APL cell line containing the t(15;17). RA treatment represses NB4 cell growth and induces a myeloid phentoype. Full length cDNAs for RAR alpha and PML were individually cloned into a CMV-driven expression vector containing the neomycin resistance gene. Surprisingly, none of the obtained stable transfectants expressed exogenous RAR alpha or PML mRNAs even when reverse transcription polymerase chain reaction (RT-PCR) detection assays were used. All clones expressed the neomycin resistance gene and were similar to parental NB4 cells in their growth and differentiation properties. An explanation explored for this lack of gene expression was that increased levels of RAR alpha or PML might suppress APL cell growth. To examine this possibility, transfection experiments were repeated using an episomal vector-based expression system containing an SV40 driven RAR alpha or PML cDNA and the hygromycin B resistance gene. A new selection strategy augmented expression of the desired cDNAs. A control episomal vector lacked a cDNA insert. Following electroporation and selection, exogenous RAR alpha expression was obtained. Compared to controls, the growth of these transfectants was markedly inhibited before and after RA-treatment and these cells more prominently induced myeloid maturation markers. In contrast, exogenous PML expression was transient since these transfectants did not appear to propagate in culture. These findings indicate: (1) a growth disadvantage for NB4 cells having increased expression of RAR alpha or PML and (2) increased RAR alpha expression augmented RA-mediated maturation of NB4 cells. This implicates a role for RAR alpha or PML in regulating the growth or differentiation of APL cells. It is hypothesized this occurs through antagonism of PML/RAR alpha actions in these leukemic cells.

Activation of c-myb is an early bone-marrow event in a murine model for acute promonocytic leukemia.

Insertional mutagenesis of c-myb by Moloney murine leukemia virus occurs in 100% of promonocytic leukemias (MMLS) induced by the virus. These leukemias, which resemble acute monocytic leukemia-M5 in humans are induced only in mice undergoing a peritoneal chronic inflammatory response. We have found that two leukemia-specific gag-myb mRNAs in MML provide molecular markers for detection of preleukemic cells in hematopoietic tissue in vivo. The two aberrant RNAs result from splicing of gag to either exon 3 or 4 of c-myb, depending on the site of proviral integration. After reverse transcription-PCR with nested primers and hybridization with specific gag-myb junction probes, one cell, having aberrant c-myb message, could be detected in a minimum of 10(5) liver cells or 10(6) spleen or bone-marrow cells. This approach was used to examine hematopoietic tissues of mice after pristane injection to induce inflammation and virus inoculation. Cells with gag-myb mRNAs could be detected as early as 2 weeks after virus inoculation. In mice receiving both pristane and virus, there was evidence of preleukemic cells in 83% of the mice by 3 weeks after virus infection. Furthermore, 100% of the mice were positive for preleukemic cells by 8 weeks, even though only 50% of mice have been shown to succumb to MML (peak time for disease latency is 12-16 weeks). Cells with these aberrant c-myb messages were initially detected in the bone marrow, but during intermediate stages of disease development these cells disseminated to the spleen, liver, and granuloma. At preleukemic times, from 3 to 8 weeks after virus infection, a lower percentage of mice were positive in the group that did not receive pristane compared with mice in the group receiving pristane. However, at 18 weeks, 100% of the mice in the group receiving virus only had evidence of cells expressing gag-myb RNA in their spleens and/or bone marrow; it is of interest that mice inoculated with virus alone never develop MML. This approach for detecting preleukemic cells will now allow the study of mechanisms by which these preleukemic cells progress to a more transformed state and, perhaps, to a more differentiated state.

Involvement of the spleen in preleukemic development of a murine retrovirus-induced promonocytic leukemia.

An acute myeloid leukemia can result from the inoculation of Moloney murine leukemia virus into BALB/c mice undergoing a 2,6,10,14-tetramethylpentadecane-induced chronic inflammatory response in the peritoneal cavity. This leukemia is ultimately observed in the peritoneal cavity as an ascites with cells infiltrating the granulomatous tissue. It has been proposed, however, that hematopoietic organs such as the spleen and bone marrow are involved in preleukemic development of Moloney murine leukemia. Therefore, to determine if the spleen plays a role in this development, mice were splenectomized at various times relative to virus inoculation. When splenectomies were performed 3 days before and 2, 4, 6, and 8 weeks after virus inoculation there was, in all cases, a decreased death rate compared to sham-splenectomized controls. The greatest difference in death rate due to promonocytic leukemia was observed when mice were splenectomized at 4 weeks after virus inoculation. The decrease in disease incidence observed as a result of splenectomy was not caused by decreased virus spread in hematopoietic organs or an alteration in the profile of the cellular infiltrate in the granuloma. It was found, however, that the spleens of 2,6,10,14-tetramethylpentadecane-treated mice, relative to those of normal mice, have a significantly increased number of granulocyte-macrophage colony-forming cells and a slightly increased number of multipotential colony-forming cells. These observations suggest that a population of target cells for transformation, consisting of granulocyte-macrophage precursor cells, may reside in the spleen. Alternatively, partially transformed cells may reside temporarily in the spleen during the developmental stages of the disease process.

Technical aspects of lymphokine-activated killer cell production.

Adoptive immunotherapy is a novel approach to treating patients with cancer, utilizing as therapy a patient's own peripheral blood lymphocytes that have been activated by incubation with interleukin-2 (IL-2). These cells develop the ability to mediate tumor regression in vivo and are referred to as lymphokine-activated killer (LAK) cells. The production of LAK cells is a complex and labor-intensive process. Lymphocytes are collected by continuous-flow centrifugation, purified on Ficoll-Hypaque (FH) density gradients, incubated in vitro with IL-2, and then harvested for infusion into the patient. An automated approach to LAK cell generation has been developed using the Fenwal CS-3000 cell separator and polyolefin PL-732 blood storage bags. Lymphocyte concentrates (LC) containing 6.5 x 10(9) mononuclear cells per pack were obtained using standard leukapheresis techniques. Disposable apheresis kits were then modified to allow the LC to be pumped into the separation chamber along with a counter-centrifugal flow of saline, removing the platelets and plasma by elutriation. The remaining cells were underlaid with FH, displacing the lymphocytes into a collection bag, where they were washed and concentrated. Mean leukocyte recovery was 59.2% (99.9% lymphocytes, n = 14). The final product contained 6.7% of the initial platelets and had a hematocrit of less than 1%.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of serum-free culture medium in the generation of lymphokine activated killer cells.

Lymphokine activated killer (LAK) cells administered in conjunction with recombinant interleukin-2 can mediate the regression of metastatic tumor in some patients with advanced cancer. In these trials LAK cells were activated in medium containing 2% human type A or AB serum. We have found three commercially available, serum-free culture media which allow development of in vitro LAK activity by human peripheral blood lymphocytes. They are AIMV (Gibco), MASF-3 (Whitaker-MA Bioproducts) and HB-104 (Dupont). If 2-mercaptoethanol was added to these culture media they were also capable of generating murine LAK cells which were effective in reducing pulmonary metastases in the murine MCA-106 model. Although LAK cells generated in these media have not been tested in humans yet, potentially they could provide a safe, unlimited and less expensive source of culture fluid for generating the large numbers of LAK cells needed for human clinical trials.

Development of an automated closed system for generation of human lymphokine-activated killer (LAK) cells for use in adoptive immunotherapy.

Immunotherapy utilizing the adoptive transfer of lymphokine-activated killer (LAK) cells in conjunction with recombinant interleukin-2 (IL-2) can mediate tumor regression in some patients with advanced cancer. The activation of large numbers of LAK cells was performed in roller bottles in a research laboratory setting and required meticulous aseptic technique, at least one skilled technician per patient and one laminar flow hood per patient. To reduce the complexity and expense of LAK cell generation for human immunotherapy trials we have developed a closed-system automated procedure using a continuous flow blood cell separator. PBL were obtained by standard apheresis techniques. Platelets and plasma were elutriated using countercentrifugal flow of saline in the cell separator machine. The washed PBL were underlaid with Ficoll-Hypaque (FH) in the original separation bag. Lymphocytes were then flushed into a collection bag where they were concentrated and washed with 2 liters of saline. Mean recovery from the automated FH technique was 54.6 +/- 4.3% compared to 62.3 +/- 4.0% using manual methods in 50 ml tubes (P greater than 0.05). Cells were diluted in the collection bag with RPMI 1640 +/- 2% human AB serum and could be dispensed in an automated fashion to polyolefin bags via a sample port with 1000-1500 U/ml IL-2. After 3-4 days of culture in 5% CO2 at 37 degrees C, activated cells from the bags were harvested and washed in a closed system using the continuous flow cell separator. Cell yield from the harvest was 79.2 +/- 5.4% in the automated system compared to 64.9 +/- 5.0% in the standard procedure using manual harvest of roller bottles (P less than 0.01). Lytic capacity of the cells against fresh human tumor in a 4 h 51Cr release assay was equivalent in cells processed either by the automated or the conventional manual method. The advantages of a closed system include decreased potential for microbial contamination and reduced labor and capital equipment costs. This technique may be easily adapted for use with other cell collection and culture systems.

Use of a continuous-flow cell separator in density gradient isolation of lymphocytes.

A continuous-flow technique was developed to isolate and concentrate lymphocytes over Ficoll-Hypaque (FH) density gradients using an automated cell separator (Model CS-3000, Fenwal). Lymphocyte concentrates (LC) containing 6.51 X 10(9) mononuclear cells were obtained by standard leukapheresis techniques. Disposable apheresis kits were modified to allow the LC to be pumped into a separation chamber along with a counter-centrifugal flow of saline, removing the platelets and plasma by elutriation. The remaining cells were underlaid with 300 ml of FH, displacing the lymphocytes into a collection bag, where they were washed and concentrated. Mean leukocyte recovery was 59.2 percent (99.9% lymphocytes, n = 14). The final product contained 6.7 percent of the initial platelets and had a hematocrit of less than 1 percent. In paired studies using split leukocyte concentrates (n = 15), lymphocyte recovery obtained by the automated apheresis technique compared favorably with that obtained by standard manual FH gradients (59.8 +/- 3.4% versus 67.3 +/- 4.2%, p greater than 0.05) and platelet contamination was significantly reduced (2.7 +/- 0.5% versus 26.6 +/- 5.7% residual platelets, p less than 0.001). Equivalent lymphokine-activated killer (LAK) activity was generated from cells isolated by both manual and automated techniques. An automated continuous-flow cell separator can be used for rapid FH isolation of large numbers of lymphocytes, providing a sterile product suitable for human use.