4HPR triggers apoptosis but not differentiation in retinoid sensitive and resistant human embryonal carcinoma cells through an RARgamma independent pathway.

Retinoids signal biological effects through retinoic acid receptors (RAR) and retinoid X receptors (RXR) and their co-regulators. We previously reported that all-trans retinoic acid (RA) triggers terminal differentiation in the human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1), through an RARgamma dependent pathway. RARgamma repression in NT2/D1-R1 cells accounts for RA resistance in this line. This report finds RARgamma repression is due to selective repression of RARgamma but not RARbeta transcription in NT2/D1-R1 cells. The repression is neither due to mutations in RARgamma nor its promoter containing the RA response element. Prior work was confirmed and extended by demonstrating that an RARgamma selective agonist preferentially signals differentiation of NT2/D1 cells, while RARalpha/beta, RARbeta, RXR agonists and an RAR pan-antagonist do not even when NT2/D1 cells are treated with these retinoids at 10 microM dosages. None of these examined retinoids induced differentiation of the RA resistant NT2/D1-R1 cells. In contrast, N-(4-hydroxyphenyl)retinamide (4HPR), a reported transcriptional activator of RARgamma was shown to potently induce growth inhibition and apoptosis in both NT2/D1 and NT2/D1-R1 cells. 4HPR-induced apoptosis was unaffected by co-treatment of both cell lines with equimolar RAR antagonist. Semi-quantitative reverse transcription-polymerase chain reaction (RT - PCR) assays of total RNA from 4HPR-treated NT2/D1 and NT2/D1-R1 cells did not reveal RARgamma induction. Since 4HPR signals in RA-resistant NT2/D1-R1 cells having an RARgamma transcriptional block, these results indicate that 4HPR triggers apoptosis but not differentiation through an RARgamma independent pathway. Taken together, these findings implicate a therapeutic role for 4HPR mediated apoptosis in germ cell tumors even when a maturation block is present.

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.

Retinoic acid stimulates protein kinase A-associated G proteins during human teratocarcinoma differentiation.

Retinoic acid (RA) treatment of F9 murine teratocarcinoma (TC) cells reduces the expression of the protein kinase A (PKA)-associated G protein, G alpha i2. The present study reveals interactions between the RA and PKA pathways during differentiation of the multipotent human TC cell line NTERA-2 clone D1 (abbreviated NT2/D1) which differ from prior reports in F9 TC cells. Compared to untreated NT2/D1 cells, differentiated NT2/D1 cells expressed increased levels of G alpha s and G alpha i1,2 proteins as shown by both immunoblot analysis and cholera toxin- and pertussis toxin-induced ADP ribosylation. To further explore cooperation between these pathways during human TC differentiation, we examined the effects of cyclic adenosine monophosphate (cAMP) on RA-responsive genes and of RA treatment on the transcriptional activation of a cAMP response element (CRE). Compared to RA alone, combined treatment with RA and cAMP augmented the expression of the RA nuclear receptor-beta (RAR-beta). Also, transient transfection assays revealed that cAMP and RA cooperated to enhance CRE transcriptional activation. The cAMP-induced enhancement of RA actions in NT2/D1 cells extended to immunophenotypic changes typical of the neuronal differentiation program induced by RA. In contrast to these findings in NT2/D1 cells, prior work in F9 TC cells showed that cAMP inhibits the RA-mediated augmentation of RAR-beta expression and switches the differentiation program from visceral to parietal endoderm. Thus, unlike murine TC cells, in human NT2/D1 cells RA stimulates PKA-associated G proteins and PKA pathway activation enhances RA-mediated TC differentiation.

Differential modulation of the CD-2 and CD-3 T cell activation pathways by a monoclonal antibody to Leu-13.

MAb anti-Leu-13 reacts with a 16-kDa-interferon-responsive lymphocyte-endothelial cell surface antigen and has been demonstrated to induce lymphocyte aggregation by an undefined adhesion pathway. While anti-Leu-13 inhibits proliferation triggered by CD3 antibodies it was found to consistently augment proliferation induced by a pair of CD2 antibodies at suboptimally mitogenic concentrations. The latter mechanism of T cell activation may represent an antigen-nonspecific activation pathway requiring extensive cell-cell interaction. Proliferation induced via the CD2 pathway was very sensitive to the presence of monocytes whose inhibitory effect was reversed by indomethacin. While the potent inhibitory effect of PGE2 on proliferation induced via the CD2 pathway was weakly antagonized by anti-Leu-13, the combined effects of anti-Leu-13 and PGE2 on the CD3 pathway were additive and very inhibitory. The possibility that the Leu-13 signal reflects a mechanism by which a monocyte/macrophage-sensitive T cell activation pathway might be selectively amplified in vivo is discussed.

Increased density of ecto 5' nucleotidase antigen on leukemic T cells from patients with cutaneous T-cell lymphoma and adult T-cell leukemia/lymphoma.

Malignant CD4+ T cells in adult T-cell leukemia/lymphoma (ATL) and cutaneous T-cell lymphoma (CTCL) express a number of cell surface molecules that are upregulated on normal T cells activated by foreign antigen. In this report we describe an interesting exception to the parallel phenotypic features of activated T cells and malignant CD4+ T cells. A monoclonal antibody (MoAb; termed 27.2) that was raised to HTLV-1+, CD4+25+ leukemic T cells stained weakly 25% of peripheral T cells, including approximately 50% of CD8+ T cells and 20% of CD4+ T cells. Flow cytometry analysis indicated that the surface density of the 27.2 antigen was unchanged or diminished when normal T cells were activated by antigen. However, 3/4 Sezary cases and 4/8 cases of ATL had relatively high densities of the 27.2 antigen. Immunoprecipitation and sodium dodecylsulfate polyacrylamide gel electrophoresis of the NP-40-solubilized membranes of surface-iodinated ATL cells indicated that MoAb 27.2 reacted with a 75 Kd molecule. The size and distribution of the 27.2 antigen on T cell subsets suggested that it might be the enzyme ecto-5' nucleotidase (NT), a phosphatidylinositol-linked enzyme that catalyzes dephosphorylation of monophosphate nucleotides to their respective nucleosides. This was confirmed by demonstrating that lymphocyte ecto-5'NT activity was blocked partially and inhibited completely by preincubating cells with MoAb 27.2 for 1 hour at 4 degrees C and 24 hours at 37 degrees C, respectively. When used with a second MoAb (27.1) to a novel T cell activation antigen found on all CTCL and ATL leukemias examined, 27.2 was found to discriminate between normal and leukemic T cells in two patients with ATL. These studies suggest that ecto-5'NT has diagnostic value in T cell malignancies and may be aberrantly expressed in some cases of ATL and CTCL.

Molecular analysis of the serologically defined HLA-Aw19 antigens. A genetically distinct family of HLA-A antigens comprising A29, A31, A32, and Aw33, but probably not A30.

The HLA-Aw19 complex consists of a number of serologically cross-reactive Ag (i.e., A29, A30, A31, A32, and Aw33) which exhibit an epitope shared by HLA-B and -C proteins. To investigate the structural basis for these serologic cross-reactivities, we have cloned and determined the nucleotide sequences for A30, A31, and Aw33, and compared the predicted amino acid sequences with those already available for A29, A32, and other class I allelic products. All alleles of the Aw19 group contained A-locus-specific sequences, exhibiting "A-ness." The structural similarities between Aw19 polypeptides were found in the alpha 1 and alpha 2 domains, where shared amino acid residues were identified that correlated with observed serological reactivity patterns. Seven Aw19-specific nucleotides were found. Two of these were silent substitutions, but the remaining five resulted in Aw19-specific amino acid residues. Each of the HLA-A alleles can be classified into one of the five serologically cross-reacting groups. In the Aw19 group, the alleles A29, A31, A32, and Aw33 are closely related serologically as well as genetically whereas A30 probably belongs to the A1/A3/A11 group. The similarity between A30 and the other Aw19 alleles may have resulted from two independent gene conversions affecting exons 2 and 3. Additional mutations or gene conversion-like events in A30 were also noted. It is postulated that gene conversions have played a significant role in the divergence of the Aw19 alleles. However, each serologically cross-reactive Aw19 allotype appears to have arisen directly from a common ancestral allele. A30 was the only exception, and this allele may represent an unusual allotype, which is subject to a high rate of genetic changes, as is seen in the H-2Kb gene of the mouse.